Biomedical subjects

C Labrie

Publications and source records attributed to C Labrie.

At least 37 records · Page 2Linked to original sources

PubMed2000

Intracrinology: role of the family of 17 beta-hydroxysteroid dehydrogenases in human physiology and disease.

In women and men, an important proportion of estrogens and androgens are synthesized locally at their site of action in peripheral target tissues. This new field of endocrinology has been called intracrinology. In postmenopausal women, 100% of active sex steroids are synthesized in peripheral target tissues from inactive steroid precursors while, in adult men, approximately 50% of androgens are made locally in intracrine target tissues. The last and key step in the formation of all estrogens and androgens is catalyzed by members of the family of 17beta-hydroxysteroid dehydrogenases (17 beta-HSDs) while different 17 beta-HSDs inactivate these steroids in the same cell where synthesis takes place. To date, seven human 17 beta-HSDs have been cloned, sequenced and characterized. The 17 beta-HSDs provide each cell with the means of precisely controlling the intracellular concentration of each sex steroid according to local needs.

17-Hydroxysteroid Dehydrogenases↗

PubMed2000

Localization of oestrogen receptor alpha, oestrogen receptor beta and androgen receptors in the rat reproductive organs.

There is now evidence that oestrogens and androgens can influence male and female reproductive systems. In order to accurately identify the sites of action of oestrogens and androgens, we have proceeded to the histological localization of the two oestrogen receptor (ER) subtypes, ERalpha and ERbeta, and the androgen receptor (AR) in the reproductive tissues of adult rats of both sexes. AR was detected by immunocytochemistry, while ERalpha and ERbeta were localized by both immunocytochemistry and in situ hybridization. In the pituitary gland of animals of both sexes, ERalpha was found in the majority of nuclei of secretory cells in the anterior pituitary. The intermediate and posterior lobes did not show any staining. ERbeta was not found to be expressed in any of the pituitary lobes. Using AR antibodies, nuclear staining was detected in about 50% of secretory cells of the anterior lobe, the intermediate and posterior lobes being completely unstained. In the testis, ERalpha was localized in nuclei of Leydig cells as well as in round spermatocytes and spermatids, while ERbeta could only be detected in Sertoli cell nuclei. AR immunoreactivity was found in nuclei of Sertoli, peritubular myoid and Leydig cells. In the prostate, ERbeta was observed in epithelial cells of tubulo-alveoli, while the stroma was unlabelled. ERalpha was not found to be expressed in any prostate cells. In the prostate, AR was detected in nuclei of epithelial, stromal and endothelial cells. In seminal vesicles, staining of ERalpha was found in nuclei of epithelial and stromal cells. Similar findings were observed using AR antibodies. While ERbeta mRNA could not be detected by in situ hybridization, weak staining for ERbeta was localized in epithelial cells of seminal vesicles. In the ovary, both ERalpha and ERbeta were found to be expressed. ERbeta mRNA was found in granulosa cells of growing follicles, while ERalpha was present in theca cells, interstitial gland cells and germinal epithelium. AR immunoreactivity was detected in granulosa cell nuclei in growing follicles and also in scattered interstitial cells. In the oviduct and uterus, ERalpha was observed in nuclei of epithelial cells as well as of stromal and muscle cells. Similarly, AR immunoreactivity was present in nuclei of epithelial cells, stromal and muscle cells in both the oviduct and uterus. ERbeta was not detected in the oviduct and uterus. The present findings indicate a cell-specific localization of ERalpha, ERbeta and AR in reproductive tissues in rats of both sexes. By establishing the precise sites of action of oestrogens and androgens they contribute to a better understanding of the respective role of these steroids in reproduction function.

Animals↗

PubMed2000

Exclusive androgenic effect of dehydroepiandrosterone in sebaceous glands of rat skin.

In order to analyze the hormonal effects of dehydroepiandrosterone (DHEA) in skin sebaceous glands, the precursor steroid was administered to ovariectomized (OVX) female Sprague-Dawley rats at a dose of 30 mg applied on the dorsal skin, twice daily, for 3, 6 and 12 months. In a parallel experiment, female OVX rats were treated with DHEA at the same daily percutaneous dose of 30 mg, alone or in combination with the antiandrogen Flutamide or the pure antiestrogen EM-800, for 12 months, in order to determine the androgenic and/or estrogenic components of DHEA action. Treatment of female OVX rats with DHEA resulted in a similar mild to moderate hyperplasia of the sebaceous glands of both dorsal (site of application) and ventral skin, as illustrated by an increase in the number and size of the acini. The above-indicated effects were observed at all time intervals studied, beginning at 3 months of treatment, and they were not further increased after longer term administration of DHEA (for 6 and 12 months). The addition of Flutamide to DHEA treatment completely prevented the DHEA-induced changes in the sebaceous glands, whereas the antiestrogen EM-800 had no effect. The present data indicate an exclusive androgenic stimulatory action of DHEA on the sebaceous glands, thus pointing out the importance of local intracrine DHEA transformation into androgens for skin anatomical integrity and function, while showing that estrogens, if active in rat skin, do not originate from DHEA.

Administration, Cutaneous↗

PubMed1999

Effects of the antiestrogen EM-800 (SCH 57050) and cyclophosphamide alone and in combination on growth of human ZR-75-1 breast cancer xenografts in nude mice.

Human breast cancer proliferates as heterogeneous cell populations that exhibit different sensitivities to therapeutic agents. A logical approach to control these different cancer cell populations is the use of combined treatment with agents that block cell proliferation or induce apoptosis via different mechanisms. We therefore investigated the effect of treatment with the novel pure antiestrogen EM-800, alone or in combination with chemotherapy, on the growth of ZR-75-1 human breast tumors in nude mice, a well-recognized model of human breast cancer. Mice bearing estrone-releasing silastic implants as estrogenic stimulus received EM-800 or cyclophosphamide alone or in combination for 227 days. Cyclophosphamide (256 mg/kg/2 weeks) was administered by i.p. injection in 64 mg/kg fractions over 4 consecutive days with repetition of the cycle every 14 days. EM-800 was administered p.o. once daily at the maximally effective dose of 300 microg/mouse. After 227 days of treatment, average tumor size in mice receiving estrone alone was 192% higher than pretreatment. The average tumor size of mice treated with chemotherapy was reduced by 47%, whereas on the other hand, EM-800 caused a 81% decrease of the value of the same parameter. The combined treatment (EM-800 + cyclophosphamide), on the other hand, resulted in a 95% decrease in tumor size compared with control estrogen alone. In fact, EM-800 alone decreased tumor size to 55% of the value at the start of treatment, whereas the addition of cyclophosphamide to the antiestrogen further decreased tumor size to as low as 15% of the pretreatment value. The combination of EM-800 and cyclophosphamide resulted in 95% of complete or partial responses compared with 61 and 27% with EM-800 and cyclophosphamide alone, respectively. In fact, in the combination therapy group, only one tumor remained stable, while 17 regressed >50% and four disappeared. It is noteworthy that no tumor progressed with EM-800 alone or in combination with cyclophosphamide. The present data show, for the first time, that the addition of cyclophosphamide to a pure antiestrogen used at a maximal dose causes a more potent inhibition of human breast tumor growth, thus suggesting that combined treatment using a maximal dose of a pure antiestrogen and a chemotherapeutic agent(s), two classes of compounds having different mechanisms of action, could further improve breast cancer therapy above the results achieved with a potent and pure antiestrogen alone in estrogen-sensitive breast cancer.

Animals↗

PubMed1999

Effect of combined treatment with the pure antiestrogen EM-800 and radiotherapy on the growth of human ZR-75-1 breast cancer xenografts in nude mice.

Human breast tumors are usually composed of heterogeneous cell populations that exhibit different sensitivities to therapeutic agents. We therefore investigated the effect of treatment with various regimens of the novel pure antiestrogen EM-800, alone or in combination with external beam radiation therapy, on the growth of human ZR-75-1 xenografts in athymic mice. The animals received a maximal dose of EM-800 (300 microg, p.o.) and/or radiotherapy at the dose of 10 Gy. 2.5 Gy fractions were administered over a 9-day period in four sessions of 13.7 min each (250-kilovolt Siemens with 2-mm aluminum filtration at 90 cm from the source origin). EM-800 was administered p.o. once daily, whereas radiotherapy was repeated every 35 days. Tumor size was expressed as a percentage of the initial tumor size, which was assigned a value of 100%. Average tumor size increased by 514% in ovariectomized mice supplemented with estrone alone for 259 days compared with the pretreatment value. Treatment with radiotherapy or EM-800 alone resulted in 11 and 73% decreases in mean tumor size, respectively, whereas combined treatment given simultaneously at the beginning caused a dramatic 98% decrease in tumor size. The start of radiotherapy on day 35 in EM-800-treated mice, or conversely, the start of EM-800 in irradiated mice at the 35-day time interval, resulted in somewhat lower, 88% and 95%, decreases in tumor size, respectively. In animals receiving EM-800 alone, 40% of tumors disappeared, thus indicating a cytotoxic effect caused by the estrogen blockade achieved with the pure antiestrogen. Eighty-six % of the original tumors disappeared under continuous combined treatment. Most importantly, no tumor reappeared under estrogenic stimulation after stopping treatment, thus indicating cure of 86% of the tumors in the group of animals who received the combination therapy. The present data indicate that combined treatment with EM-800 and radiotherapy yields a faster response, a greater decrease in tumor size, and a higher percentage of complete responses or tumor disappearance (cure) than either treatment used alone. The present data also suggest that maximal benefits are achieved when the pure antiestrogen is administered continuously, starting at the same time as radiation therapy and continued without interruption as adjuvant therapy. The present data also clearly show that efficient blockade of estrogens with a potent and pure antiestrogen is not only cytostatic but is cytotoxic and can lead to the disappearance of an important proportion of tumors or cure.

Animals↗

PubMed1999

Expression profile of agonistic Smads in human breast cancer cells: absence of regulation by estrogens.

Transforming growth factor-beta1 (TGF-beta1) is a cytokine expressed by mammary cells. While TGF-beta1 can inhibit the proliferation of human breast cancer cells, many cell lines are unresponsive to it. To shed light on the mechanisms underlying resistance to TGF-beta1, we examined expression of the mediators of TGF-beta1 signaling in the mammary carcinoma cell lines MCF-7, T47D, ZR-75-1, BT-20, MDA-MB-231 and MDA-MB-468. The levels of mRNA encoding Smad2, 3 and 4 as well as the type II (TbetaRII) and type I (TbetaRI) membrane receptors were determined by Northern analysis and/or ribonuclease protection assays. Smad2 and Smad3 mRNAs were detected in all 6 cell lines examined, whereas Smad4 mRNA was not detected in MDA-MB-468 cells, which are known to harbor a homozygous deletion of the Smad4 gene. TbetaRI was expressed in all 6 cell lines, whereas TbetaRII was not detected in ZR-75-1 and T47D cells. Of the cell lines tested, only MCF-7 cells were growth-inhibited by TGF-beta1. In contrast, only MDA-MB-231 cells showed induction of the PAI-1 promotor in response to TGF-beta1. We also examined the regulation of Smad mRNA expression by estrogens and androgens in ZR-75-1 cells. Neither estradiol nor dihydrotestosterone affected Smad2, 3 or 4 mRNA levels in ZR-75-1 cells. These results indicate that the lack of response to TGF-beta1 in the breast cancer cell lines examined can be attributed to the absence of either TbetaRII or the Smad4 gene product. Moreover, we show that the proliferative and transcriptional responses to TGF-beta1 are dissociable and that Smad expression is not regulated by sex steroids in ZR-75-1 cells.

Breast Neoplasms↗

PubMed1999

Androgens down-regulate bcl-2 protooncogene expression in ZR-75-1 human breast cancer cells.

Although a large proportion of primary human breast cancers express the androgen receptor, and treatment with androgens exerts beneficial effects in women with breast cancer, the role and especially the mechanism of action of androgens in breast cancer development and growth are not well understood. The potential effect of androgens on bcl-2 protooncogene expression was investigated in a human breast cancer cell line whose proliferation is known to be inhibited by androgens. The estrogen-responsive ZR-75-1 cells were grown in the presence or absence of 5alpha-dihydrotestosterone (DHT), alone or in combination with 17beta-estradiol. DHT caused a marked down-regulation of Bcl-2 protein and messenger RNA levels in both the presence and absence of 17beta-estradiol. The inhibitory effect of DHT was completely prevented by coincubation with the pure antiandrogen hydroxyflutamide. The present data indicate that androgens can down-regulate bcl-2 protooncogene levels via an androgen receptor-mediated mechanism, thus providing a novel mechanism for their known inhibitory effect on breast cancer cell growth.

Androgen Antagonists↗

PubMed1999

Identification and cloning of a novel androgen-responsive gene, uridine diphosphoglucose dehydrogenase, in human breast cancer cells.

Androgens inhibit the growth of breast cancer cells, but the mechanism of androgen-induced growth inhibition has not yet been elucidated, and few androgen-responsive genes have been identified. We, therefore, used differential display PCR to identify novel androgen-responsive genes in ZR-75-1 human breast cancer cells. The human UDP-glucose dehydrogenase gene (UDPGDH), which was not known to be androgen regulated, was detected and cloned by complementary DNA library screening. The UDPGDH open reading frame codes for a protein of 494 amino acids that migrates at an apparent molecular mass of approximately 54 kDa. Northern blot analysis revealed the existence of two messenger RNA species of approximately 3.5 and 2.7 kb in all of the human breast cancer cell lines examined. The major UDPGDH transcript was induced rapidly (within 6 h) by dihydrotestosterone in ZR-75-1 cells, and a maximal 13-fold induction was observed after 24 h of treatment. The increase in UDPGDH messenger RNA was completely prevented by coincubation with the pure antiandrogen hydroxyflutamide, but not by cycloheximide, indicating that UDPGDH is directly regulated by the androgen receptor. As UDPGDH is required for the production of uridine 5'-diphosphoglucuronic acid, a substrate for the steroid-conjugating uridine diphospho-glucuronosyltransferase enzymes, up-regulation of UDPGDH expression by androgens might play an important role in the control of sex steroid inactivation via glucuronidation in breast cancer cells.

Amino Acid Sequence↗

PubMed1998

Expression and neuropeptidergic characterization of estrogen receptors (ERalpha and ERbeta) throughout the rat brain: anatomical evidence of distinct roles of each subtype.

The recent cloning of a second estrogen receptor (ER) provided a new tool to investigate and clarify how estrogens are capable of communicating with the brain and influence gene expression and neural function. The purpose of the present study was to define the neuroanatomical organization of each receptor subtype using a side-by-side approach and to characterize the cellular population (s) expressing the ERbeta transcript in the endocrine hypothalamus using immunohistochemistry combined with in situ hybridization. Axonal transport inhibition was accomplished to cause neuropeptide accumulation into the cytoplasm and thus facilitate the detection of all positive luteinizing hormone-releasing hormone (LHRH), corticotropin-releasing factor (CRF), vasopressin (AVP), oxytocin (OT), gastrin-related peptide (GRP), and enkephalin (ENK) neurons. The genes encoding either ERalpha or -beta were expressed in numerous limbic-associated structures, and fine differences were found in terms of intensity and positive signal. Such phenomenon is best represented by the bed nucleus of the stria terminalis (BnST) and preoptic area/anterior hypothalamus, where the expression pattern of both transcripts differed across subnuclei. The novel ER was also found to be expressed quite exclusively in other hypothalamic nuclei, including the supraoptic (SON) and selective compartments (magnocellular and autonomic divisions) of the paraventricular nucleus (PVN). A high percentage of the ERbeta-expressing neurons located in the ventro- and dorsomedial PVN are of OT type; 40% of the OT-ir cells forming the medial magnocellular and ventromedial parvocellular PVN showed a clear hybridization signal for ERbeta mRNA, whereas a lower percentage (15-20%) of OT neurons were positive in the caudal parvocellular PVN and no double-labeled cells were found in the rostral PVN and other regions of the brain with the exception of the SON. Very few AVP-ir neurons expressing ERbeta transcript were found throughout the rat brain, although the medial PVN displayed some scattered double-labeled cells (<5%). Quite interestingly, the large majority of the ERbeta-positive cells in the caudal PVN were colocalized within CRF-ir perikarya. Indeed, more than 60-80% of the CRF-containing cells located in the caudolateral division of the parvocellular PVN exhibited a positive hybridization signal for ERbeta mRNA, whereas very few (<5%) neuroendocrine CRF-ir parvocellular neurons of the medial PVN expressed the gene encoding ERbeta. A small percentage of ERbeta-expressing cells in the dorsocaudal and ventromedial zones of the parvocellular PVN were also ENK positive. The ventral zone of the medial parvocellular PVN also displayed GRP-ir neurons, but no convincing hybridization signal for ERbeta was detected in this neuronal population. Finally, as previously described for the gene encoding the classic ER, LHRH neurons of both intact and colchicine-pretreated animals did not express the novel estrogen receptor. This study shows a differential pattern of expression of both receptors in the brain of intact rats and that ERbeta is expressed at various levels in distinct neuropeptidergic populations, including OT, CRF, and ENK. The influence of estrogen in mediating genomic and neuronal responses may therefore take place within these specific cellular groups in the brains of cycling as well as intact male mammals.

Animals↗

PubMed1998

Structure of the gene encoding the human cyclin-dependent kinase inhibitor p18 and mutational analysis in breast cancer.

The cyclin-dependent kinase (CDK) inhibitor p18 blocks progression of the cell cycle by associating with the cyclin D-dependent kinases CDK6 and CDK4. To better understand the regulation of p18 gene expression, we isolated full-length cDNA clones from a human BT-20 breast cancer cell cDNA library. These clones were then used to isolate the human gene from a human genomic DNA library. The human p18 gene spans at least 7.5 kb and is composed of three exons, two of which encode the p18 protein. The genomic clone we isolated contained 5 kb of putative promotor sequence which directed expression of the luciferase reporter gene in transient transfection experiments. The longest cDNA that we isolated from BT-20 cells contained 2103 nucleotides which corresponds to the size of the major RNA transcript detected by Northern analysis in these cells. Transcription start sites mapping to the 5' end of the putative full-length cDNA were identified by ribonuclease protection assays. A novel polymorphism was identified in the 3' untranslated region of BT-20 cell cDNA clones that contained the previously described codon 72 mutation. The codon 72 mutation was also detected in 3 of 35 breast tumors analyzed using a mismatch PCR/RFLP strategy.

Amino Acid Sequence↗

PubMed1998

Effect of dehydroepiandrosterone and the antiestrogen EM-800 on growth of human ZR-75-1 breast cancer xenografts.

BACKGROUND: In the mammary gland, androgens are formed from the precursor steroid dehydroepiandrosterone (DHEA). Clinical evidence indicates that androgens have inhibitory effects on breast cancer. Estrogens, on the other hand, stimulate the development and growth of breast cancer. We studied the effect of DHEA alone or in combination with the newly described pure antiestrogen EM-800 on the growth of subcutaneous tumor xenografts formed by the human breast cancer cell line ZR-75-1 in ovariectomized nude mice. METHODS: Immediately after ovariectomy, mice received daily subcutaneous injections of 0.5 microg estrone (E1) (an estrogenic hormone). EM-800 (15, 50, or 100 microg) was given orally once daily. DHEA was administered percutaneously twice daily (total dose of 0.3, 1.0, or 3.0 mg) to the dorsal skin either alone or in combination with a 15-microg daily oral dose of EM-800. Changes in tumor size in response to the treatments (in relation to measurements made on the first day of treatment) were assessed periodically. At the end of the experiments, tumors were dissected and weighed. RESULTS: A 9.4-fold increase in tumor size in 9.5 months was observed in ovariectomized mice receiving E1 alone. Administration of 15, 50, or 100 microg EM-800 in E1-supplemented mice led to inhibitions of 87.5%, 93.5%, and 94.0% in tumor size, respectively. DHEA, on the other hand, at doses of 0.3, 1.0, or 3.0 mg inhibited terminal tumor size by 50.4%, 76.8%, and 80.0%, respectively. Comparable inhibitions in tumor size were obtained with a daily 15-microg oral dose of EM-800 with or without different doses of percutaneous DHEA. CONCLUSIONS: DHEA and EM-800 independently suppressed the growth of E1-stimulated ZR-75-1 xenograft tumors in nude mice. Administration of DHEA at the defined doses did not alter the inhibitory effect of EM-800.

Administration, Cutaneous↗

PubMed1998

Comparison of the effects of the antiestrogens EM-800 and tamoxifen on the growth of human breast ZR-75-1 cancer xenografts in nude mice.

Although estrone supplementation in ovariectomized (OVX) nude mice bearing ZR-75-1 xenografts caused a 365% increase in average tumor size during the 4-month treatment period, administration of the antiestrogen EM-800 at the daily oral doses of 50, 150, or 400 microg completely prevented estrogen-stimulated tumor growth. At the same doses of tamoxifen, tumor size was inhibited to 189, 117, and 120% above pretreatment values. However, when EM-800 (150 microg/day) was added to the daily 150- and 400-microg doses of tamoxifen, final tumor size was decreased further to 12 and 38% above pretreatment values, respectively. EM-800 (400 microg daily) administered to estrone-supplemented OVX mice caused complete, partial, and stable responses in 11, 22, and 49% of estrone-stimulated tumors, respectively, whereas 19% (7 of 37) progressed. At the same dose of tamoxifen, the corresponding responses were 3% (complete response), 3% (partial response), and 25% (no change), whereas 69% (22 of 32) of tumors progressed. In the absence of estrone supplementation, tamoxifen (400 microg) alone administered to OVX mice stimulated tumor growth to 161% compared with initial size whereas the same dose of EM-800 reduced tumor size by 55%, a value superimposable to that observed in OVX control animals. The agonistic effect of tamoxifen is thus illustrated by the observation that 73% of tumors progressed when tamoxifen was administered alone to OVX animals whereas no tumor progressed with EM-800. The present data strongly suggest that at least part of the initial lack of response and resistance to tamoxifen during tamoxifen treatment in women is due to the estrogenic activity of this compound, whereas the new antiestrogen EM-800 exerts pure antagonistic action.

Analysis of Variance↗

PubMed1998

Prevention of development of dimethylbenz(a)anthracene (DMBA)-induced mammary tumors in the rat by the new nonsteroidal antiestrogen EM-800 (SCH57050).

The effect of EM-800, a new non-steroidal antiestrogen having pure antiestrogenic activity, was studied on chemical carcinogenesis induced by dimethylbenz(a)anthracene (DMBA) as well as on serum lipids and bone mass in the rat. Treatment with EM-800 orally, once daily, for 282 days (9 months), starting 3 days before DMBA administration, decreased the incidence of tumors from 95% in control animals to 60% (p < 0.01), 38% (p < 0.01), and 28% (p < 0.01) at the daily doses of 25 microg, 75 microg, and 250 microg, respectively. The average number of tumors per animal decreased from 4.5 +/- 0.5 tumors in the control group to 0.9 +/- 0.2 (p < 0.01), 0.5 +/-0.2 (p < 0.01), and 0.3 +/- 0.1 (p < 0.01) tumors in the rats treated with the above-indicated doses of the anti-estrogen. In addition, treatment with the increasing doses of EM-800 reduced serum cholesterol levels to 64%, 56%, and 48% of control, while serum triglycerides decreased to 31%, 28%, and 30% of control. Bone mineral content (BMC) and bone mineral density (BMD) of total skeleton, femur, and lumbar spine were not significantly affected following 282 days of treatment with EM-800. However, treatment with EM-800 inhibited the urinary ratio of hydroxyproline to creatinine (HP/Cr) from 14.0 +/- 3.90 micromol/mmol in controls to 7.6 +/-0.8 (p < 0.05), 6.8 +/- 0.8 (p < 0.01), and 6.8 +/- 1.1 (p < 0.01) micromol/mmol, respectively, while the same treatment had no effect on serum total alkaline phosphatase (tALP) activity or urinary calcium and phosphorus excretion. The 25 microg, 75 microg, and 250 microg daily doses of EM-800 inhibited uterine weight by 35% (p < 0.01), 62% (p < 0.01), and 66% (p < 0.01), while vaginal weight was reduced by 8% (p < 0.05), 30% (p < 0.01), and 38% (p < 0.01), respectively. In agreement with the 27% increment (p < 0.05) in ovarian weight at the highest anti-estrogen dose used, serum androstenedione (p < 0.05), androst-5-ene-3beta,17beta-diol (p < 0.01), testosterone (p < 0.05), and estradiol (p < 0.01) levels were increased. The present data show that EM-800 prevents the development of DMBA-induced mammary tumors while simultaneously inhibiting uterine and vaginal weight, reducing serum cholesterol and triglyceride levels, and having no adverse effect on bone mass following 9 months of treatment in the rat.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1998

EM-800, a novel antiestrogen, acts as a pure antagonist of the transcriptional functions of estrogen receptors alpha and beta.

Estrogens act as potent mitogens in a large number of breast cancers, and the use of estrogen receptor (ER) antagonists is, therefore, considered the endocrine therapy of choice in the management of this disease. We describe the molecular properties of EM-652, the active metabolite of EM-800, a novel nonsteroidal antiestrogen compound, on the transcriptional functions of ER alpha and ER beta. Using RT-PCR, we show that ER alpha and ER beta are expressed in mouse mammary glands, suggesting that both receptors should be considered putative targets for antiestrogen action in the breast. In cotransfection assays using a synthetic estrogen-responsive promoter, EM-652 shows no agonistic activity on ER alpha and ER beta transcriptional function and blocks the estradiol (E2)-mediated activation of both ER alpha and ER beta. EM-652 is also very effective in abrogating E2-stimulated ER alpha and ER beta trans-activation of the pS2 promoter in HeLa cells. EM-652 does not alter binding of ER alpha and ER beta to DNA. The Ras-mediated induction of ER alpha and ER beta transcriptional activity in the presence of E2 is also completely abolished by EM-652. In addition, EM-652 blocks the E2-dependent activation of ER alpha and ER beta by the steroid hormone receptor coactivator-1 as well as the in vitro interaction between SRC-1 and the ligand-binding domains of both ERs. These results demonstrate that the novel antiestrogen EM-800 fully impedes AF-1 and AF-2 activities of ER alpha and ER beta and can, therefore, be considered a potent and pure antagonist of both ER subtypes.

Animals↗

PubMed1998

Almost exclusive androgenic action of dehydroepiandrosterone in the rat mammary gland.

To determine the relative role of the androgenic and/or estrogenic components of the action of dehydroepiandrosterone (DHEA) on the histomorphology and structure of the rat mammary gland, ovariectomized (OVX) female animals received DHEA administered alone or in combination with the pure antiandrogen flutamide or the pure antiestrogen EM-800 for 12 months. We have also evaluated the effect of estradiol (E2) and dihydrotestosterone constantly released from SILASTIC brand silicon implants as well as medroxyprogesterone acetate released from poly(lactide-co-glycolide) microspheres. While 1-yr OVX resulted in a severe atrophy of the mammary gland, treatment of OVX animals with DHEA stimulated lobuloalveolar and ductal growth, as well as the secretory activity of the acinar cells, thus resulting in a lobuloalveolar type of development of the mammary gland. The addition of FLU to DHEA almost completely prevented the stimulatory effect observed with DHEA alone, whereas addition of the antiestrogen EM-800 had no significant effect on the action of DHEA on the mammary gland. At the doses used, medroxyprogesterone acetate and dihydrotestosterone also stimulated ductal and alveolar development, although to a lesser degree than that achieved with DHEA. The stimulatory effect of estradiol was mainly expressed on ductal growth with a smaller stimulatory effect on lobuloalveolar development. The above-indicated stimulatory effects on lobuloalveolar development were also reflected in significant increases of the total and parenchymal gland surface areas of the mammary gland. The present study shows that androgens induce a marked lobuloalveolar type of development of the mammary gland in the rat. Moreover, these data indicate the highly predominant or almost exclusive androgenic component in the potent stimulatory action of DHEA on the histomorphology and structure of the rat mammary gland. In fact, blockade of the potential estrogenic component of DHEA action by EM-800 did not affect the stimulatory action of DHEA on mammary gland histomorphology, whereas the antiandrogen FLU almost completely blocked the effect of DHEA.

Androgen Antagonists↗

PubMed1998

Comparison of the effects of the new orally active antiestrogen EM-800 with ICI 182 780 and toremifene on estrogen-sensitive parameters in the ovariectomized mouse.

The nonsteroidal antiestrogen EM-800 is approximately 10-fold more potent than ICI 182 780, the most potent known steroidal antiestrogen, at inhibiting estrone-stimulated uterine weight in ovariectomized mice (half-maximal inhibitory daily s.c. doses of 0.2 and 2.0 microg, respectively). At maximal doses, however, both compounds lead to a similar maximal 90% inhibition of estrone-stimulated uterine weight. A 10-fold higher activity of EM-800 compared with ICI 182 780 was also observed on estrone-stimulated vaginal weight, with maximal inhibitions of 96% and 90%, respectively, achieved by the two compounds. In addition, EM-800 injected s.c. or administered orally led to a marked loss of uterine and vaginal estrogen receptor levels measured by binding assay, whereas ICI 182 780 exerted no inhibitory effect on this parameter under the experimental conditions used. Comparable effects were observed when estrogen receptor protein levels were measured by enzyme immunoassay. After oral administration, EM-800 exerted maximal 83% and 88% inhibitions of uterine and vaginal weight, respectively, whereas maximal inhibitions limited to 51% and 67% were achieved with toremifene. This limited inhibition by toremifene of the stimulatory effect of estrone on uterine and vaginal weight is probably due to the intrinsic estrogenic activity of the compound. The present data also show that the steroidal antiestrogen ICI 182 780 has less than 3% the activity of EM-800 when administered by the oral route. In fact, EM-800 administered orally is 2- to 3-fold more potent than ICI 182 780 injected s.c.

Animals↗

PubMed1998

Effect of twenty-four-week treatment with the antiestrogen EM-800 on estrogen-sensitive parameters in intact and ovariectomized mice.

Treatment with the antiestrogen EM-800, at the daily oral dose of 3 microg, 10 microg, 30 microg, or 100 microg for 24 weeks, caused a marked inhibition of uterine and vaginal weight in both intact and ovariectomized mice. Maximal 64% and 41% inhibitions of uterine weight were achieved in intact and ovariectomized animals, respectively. Similar inhibitory effects of EM-800 were observed on vaginal weight with maximal inhibitions of 71% and 35%, in intact and ovariectomized animals, respectively. The pure antiestrogenic activity of EM-800 on the hypothalamo-pituitary-ovarian axis is illustrated by the 76-91% increases in ovarian weight observed in intact animals treated with the 10-100 microg doses of the antiestrogen. Serum 17beta-estradiol was 93% increased at the 100 microg daily dose of EM-800, whereas serum androstenedione, testosterone, and dihydrotestosterone were 141-713% increased over control at the same dose of the antiestrogen. Serum LH was increased by treatment with EM-800 in intact animals, whereas no effect was observed on the elevated gonadotropin levels in ovariectomized animals. At all doses used in intact animals, the antiestrogen caused a complete disappearance of the glandular elements of the mammary gland, the atrophy being comparable with that observed in ovariectomized mice. The mammary gland of EM-800-treated animals was exclusively composed of an atrophied ductal system lined by atrophied epithelial cells with an absence of lobulo-glandular elements. No effect of the compound was observed on the histology of the mammary gland in ovariectomized animals, thus showing the pure antiestrogenic effect of EM-800 on the mammary gland, as shown also for the uterus, vagina, and hypothalamo-pituitary axis. At histopathology, all doses of EM-800 in intact animals led to a moderate to severe uterine and vaginal atrophy. The uterine atrophy affected both the myometrium and the endometrium. Interestingly, the uterine atrophy achieved in intact animals treated with EM-800 was greater than that observed after ovariectomy alone, thus clearly demonstrating the pure antiestrogenic activity of EM-800. The present data show the highly potent and pure antiestrogenic activity of EM-800 on all parameters measured after 6 months of treatment in both intact and ovariectomized mice, a maximal effect being reached at the daily 10 microg dose of the antiestrogen in intact animals.

Androstenedione↗

PubMed1998

Predominant androgenic component in the stimulatory effect of dehydroepiandrosterone on bone mineral density in the rat.

In order to assess the relative roles of the androgenic and/or estrogenic components in the stimulatory effect of dehydroepiandrosterone (DHEA) on bone mineral content (BMC) and density (BMD), ovariectomized (OVX) female rats received DHEA administered alone or in combination with the antiandrogen flutamide (FLU) or the antiestrogen EM-800 for 12 months. We also evaluated, for comparison, the effect of estradiol (E2) and dihydrotestosterone (DHT) constantly released by Silastic implants as well as medroxyprogesterone acetate (MPA) released from poly(lactide-co-glycolide) microspheres. Femoral BMD was decreased by 11% 1 year after OVX, but treatment of OVX animals with DHEA increased BMD to a value 8% above that of intact animals. The administration of FLU reversed by 76% the stimulatory effect of DHEA on femoral BMD and completely prevented the stimulatory effect of DHEA on total body and lumbar spine BMD. Similar results were obtained for BMC. On the other hand, treatment with the antiestrogen EM-800 did not reduce the action of DHEA on BMD or BMC. At the doses used, MPA, E2 and DHT increased femoral BMD, but to a lesser degree than observed with DHEA. Bone histomorphometry measurements were also performed. While DHEA treatment partially reversed the marked inhibitory effect of OVX on the tibial trabecular bone volume, the administration of FLU inhibited by 51% (P < 0.01) the stimulatory effect of DHEA on this parameter. The addition of EM-800 to DHEA, on the other hand, increased trabecular bone volume to a value similar to that of intact controls. DHEA administration markedly increased trabecular number while causing a marked decrease in the intertrabecular area. The above stimulatory effect of DHEA on trabecular number was reversed by 54% (P < 0.01) by the administration of FLU, which also reversed by 29% the decrease in intertrabecular area caused by DHEA administration. On the other hand, the addition of EM-800, while further decreasing the intertrabecular space achieved by DHEA treatment, also led to a further increase in trabecular number to a value not significantly different from that of intact control animals, suggesting an additional effect of EM-800 over that achieved by DHEA. Treatment with DHEA caused a 4-fold stimulation of serum alkaline phosphatase, a marker of bone formation, while the urinary excretion of hydroxyproline, a marker of bone resorption, was decreased by DHEA treatment. Treatment with DHEA and DHEA + EM-800 decreased serum cholesterol levels by 22 and 65% respectively, while the other treatments had no significant effect on this parameter. The present data indicate that the potent stimulatory effect of DHEA on bone in the rat is mainly due to the local formation of androgens in bone cells and their intracrine action in osteoblasts.

Androgen Antagonists↗