Differential effects of 17beta-estradiol and raloxifene on VSMC phenotype and expression of osteoblast-associated proteins

Several studies demonstrate an association between osteoporosis and arterial calcific disease, both of which being common in elderly women. Estradiol and raloxifene, a selective estrogen receptor modulator, prevent bone loss in postmenopausal women. Little is known regarding how these agents affect arterial calcification. The aim of this study was to determine whether or not 17beta-estradiol and raloxifene reduced vascular smooth muscle cell (VSMC) differentiation and expression of bone-associated proteins during phosphate-induced calcification in vitro. Aortic VSMC were cultured from adult, gonadally intact, and ovariectomized (OVX) female pigs. Calcifying medium was added, and cells were treated with solvent (control), 17beta-estradiol (E(2)), or raloxifene. Extent of calcification and phenotypic expression of bone-associated proteins [matrix gla protein (MGP), osteoprotegerin (OPG), and bone sialoprotein (BSP)] were examined at 3-day intervals over 2 wk. Calcium content increased in all groups but was greater in VSMC derived from intact compared with OVX animals. E(2) reduced calcification and preserved a contractile phenotype. Expression of OPG significantly decreased with time; this decrease was significantly greater in VSMC derived from OVX compared with gonadally intact pigs. E(2) and raloxifene preserved expression of OPG only in VSMC from intact pigs. Expression of MGP increased significantly with time and was not affected by E(2) or raloxifene treatments. E(2) treatment significantly inhibited synthesis of BSP in cells from both groups. In conclusion, E(2) slows differentiation of VSMC induced by excess phosphate. Effectiveness of raloxifene to preserve expression of bone cell-associated proteins depends on the hormonal status of the tissue donor.     

High glucose blocks the effects of estradiol on human vascular cell growth: differential interaction with estradiol and raloxifene

Because diabetic women appear not to be protected by estrogen in terms of propensity to cardiovascular disease, we tested the possibility that chronic hyperglycemia modulates the effects of E(2) on vascular cell growth in vitro. Human endothelial cells (E304) and vascular smooth muscle cells (VSMC) were grown in normal glucose (5.5 mmol/l), high glucose (22 mmol/l) or high manitol (22 nmol/l; an osmotic control) for 7 days. In endothelial cells glucose per se stimulated DNA synthesis. However E(2)- (but not RAL-) stimulated [3H] thymidine incorporation was attenuated in the presence of high glucose. In parallel, E(2)-dependent MAP-kinase-kinase activity was blocked in the presence of high glucose. High glucose increased basal creatine kinase (CK) specific activity, but E(2)-stimulated CK was not significantly impaired in the presence of high glucose. In VSMC, high glucose prevented the inhibitory effect of high E(2) (but not of high RAL) concentrations on DNA synthesis. High glucose also prevented E(2)-induced MAP-kinase-kinase activity. In contrast, while high glucose augmented basal CK, the relative E(2)-induced changes were roughly equal in normal and high high glucose media. Hence, high glucose blocks several effects of E(2) on vascular cell growth, which are mediated, in part, via the MAP-kinase system and are likely contributors to E(2)'s anti-atherosclerotic properties. Since RAL's estrogen-mimetic effects on human vascular cell growth were independent of MAP-kinase activation and were not affected by hyperglycemia, the potential use of RAL to circumvent the loss of estrogen function induced by hyperglycemia and diabetes in the human vasculature should be further explored.     

Diabetes modulates differentially creatine kinase-specific activity responsiveness to estradiol-17beta and to raloxifene in rat organs

Diabetes mellitus increases the risk for CVD in women. While there is considerable evidence suggesting beneficial effects of estrogen on decreasing lipid peroxidation, atherosclerotic processes, and cardiovascular diseases, diabetes negates most estrogen protective effects as well as the skeletal protective effects of estrogens, which are not discernable in diabetic women. In the present study, we examined the in vivo effects of estradiol-17beta (E2), on creatine kinase (CK)-specific activity, in estrogen-responsive organs from healthy and diabetic rats. Healthy or diabetic (streptozotocin-induced) female rats were injected with either E2 (10-50 microg/rat) or raloxifene (Ral; 500-1,000 microg/rat). Twenty-four hours following the injection, animals were sacrificed; their organs removed and assayed for CK-specific activity. CK-specific activity in different organs [Left ventricle of heart (Lv), uterus (Ut), aorta (Ao), para uterine adipose tissue (Ad), epiphyseal cartilage (Ep), and diaphyseal bone (Di)] from healthy animals, was stimulated with increased doses of E2, with maximum at 20 microg/rat. Age-matched diabetic female rats exhibited a remarkable decreased response to E2 in all organs except Ut. In contrast, the response to Ral was not altered in diabetic rats. Similar results were observed in organs from ovariectomized female rats (Ovx), healthy or diabetic. These results support our previous in vitro findings, demonstrating that hyperglycemia decreases CK response to E2 but not to Ral in cultured human vascular and bone cells. In summary, diabetes mellitus decreases CK response to E2 but not that of Ral in skeletal and vascular tissues. The decreased response to E2 detected in organs derived from diabetic rats might be due to changes in nuclear and/or membrane estrogen receptors and/or other genomic and non-genomic pathways, as was shown in in vitro cellular models.     

Suppression by 17beta-estradiol of monocyte adhesion to vascular endothelial cells is mediated by estrogen receptors

Several observational studies have shown that estrogen replacement therapy decreases cardiovascular mortality and morbidity in postmenopausal women. However, The Women's Health Initiative (WHI) study has found that women receiving estrogen plus progestin had a significantly higher risk of breast cancer, coronary heart disease, stroke, and pulmonary embolus. In the present study, we examined whether estrogen prevents mechanisms that relate to plaque formation by inhibiting monocyte adhesion to endothelial cells. ECV304 cells, an endothelial cell line that normally expresses minimal estrogen receptor (ER)alpha, were transfected with an ERalpha expression plasmid. Treatment with tumor necrosis factor (TNF)-alpha increased expression of vascular cell adhesion molecule (VCAM)-1 mRNA, activation of nuclear factor-kappaB (NF-kappaB), and U937 cell adhesion in ECV304 cells. These effects of TNF-alpha were not significantly inhibited by pretreatment of native ECV304 cells with 17beta-estradiol (E(2)). In ECV304 cells overexpressing ERalpha, E(2) significantly inhibited the effects of TNF-alpha on NF-kappaB activation, VCAM-1 expression, and U937 cell adhesion. These findings suggest E(2) suppresses inflammatory cell adhesion to vascular endothelial cells that possess functional estrogen receptors. The mechanism of suppression may involve inhibition of NF-kappaB-mediated up-regulation of VCAM-1 expression induced by atherogenic stimuli. E(2) may prevent plaque formation, as first stage of atheroscrelosis through inhibiting adhesion monocytes to endothelial cell. Actions of estrogen replacement therapy can be assessed in terms of densities of functional ERalpha.     

Membranal effects of phytoestrogens and carboxy derivatives of phytoestrogens on human vascular and bone cells: new insights based on studies with carboxy-biochanin A

Estradiol-17beta (E2) and some phytoestrogens induce a biphasic effect on DNA synthesis in cultured human vascular smooth muscle cells (VSMC), i.e., stimulation at low concentrations and inhibition at high concentrations. These compounds also increase the specific activity of creatine kinase (CK) as well as intracellular Ca2+ concentration in both VSMC and human female-derived cultured bone cells (OBs), and stimulate ERK1/2 phosphorylation in VSMC. At least some of these effects are exerted via membranal binding sites (mER), as would appear from observations that protein-bound, membrane impermeant estrogenic complexes can mimic the effect of E2 on DNA synthesis, intracellular Ca2+ concentration and MAPK, but not on CK activity. We now extend these studies by examining the effects of a novel carboxy-derivative of biochanin A, 6-carboxy-biochanin A (cBA) in VSMC and human osteoblasts in culture. cBA increased DNA synthesis in VSMC in a dose-dependent manner and was able to maintain this effect when linked to a cell membrane impermeable protein. In VSMC both cBA and estradiol, in their free or protein-bound forms induced a steep and immediate rise in intracellular calcium. Both the free and protein-bound conjugates of cBA and estradiol increased net MAPK-kinase activity. Neither the stimulatory effect of cBA nor the inhibitory effect of estradiol on DNA synthesis in VSMC could be shown in the presence of the MAPK-kinase inhibitor UO126. The presence of membrane binding sites for both estradiol and cBA was supported by direct visualization, using fluorescence labeling of their respective protein conjugates, E2-BSA and cBA-ovalbumin. Furthermore, these presumed membrane ER for estradiol and cBA were co-localized. In cultured human osteoblasts, cBA stimulated CK activity in a dose related fashion, which paralleled the increase in CK induced by estradiol per se, confirming the estrogenic properties of cBA in human bone cells. Both the free and protein-bound forms of cBA elicited immediate and substantial increments in intracellular Ca2+, similar to, but usually larger than the responses elicited by estradiol per se. cBA also increased ERalpha and suppressed ERbeta mRNA expression in human osteoblasts. Cultured human osteoblasts also harbor membrane binding sites for protein-bound form of cG, which are co-localized with the binding sites for protein-bound estradiol. The extent to which these properties of the novel synthetic phytoestrogen derivatives may be utilized to avert human vascular and/or bone disease requires further study.     

Effects of selective estrogen receptor modulator raloxifene plus 17-beta estradiol in aorta and mammary gland of female experimental atherosclerosis rabbits and possible involvement of ERK signal transduction pathway

The present study investigated the effects of raloxifene, a second generation selective estrogen receptor modulator (SERM), plus 17-betaE2 on aortic atherosclerosis and mammary gland hyperplasia in ovariectomized, cholesterol-fed rabbits. Following 10 weeks of raloxifene, 17-betaE2, or raloxifene plus 17-betaE2 administration, serum total cholesterol, triglyceride, low density lipoprotein were significantly decreased in the drug groups compared to the placebo group. Consistent with serum lipid results, the total lesion area for each aorta of the drug groups decreased significantly as compared to the placebo group. HE staining of aorta paraffin section showed that in the drug groups the endothelial monolayer was almost continuous. While in mammary gland, HE staining of paraffin sections indicated the hyperplasia of epithelial cells (in 17-betaE2 group) was obviously inhibited in raloxifene plus 17-betaE2 group. In cultured vascular smooth muscle cell (VSMC), the results of MTT and [3H]TdR incorporation showed that the drug groups could inhibit AngII-induced proliferation of VSMC. Western blotting proved that raloxifene plus 17-betaE2 inhibited the expression of phosphorylated ERK protein, similar to 17-betaE2 but different from raloxifene. This effect was inhibited by PD98059 (inhibitor of MAPK) or ICI182780 (ER antagonist). In conclusion, this study suggests that SERM raloxifene plus 17-betaE2 improves the lipid metabolism and relieves the aorta changes of female experimental atherosclerosis rabbits, which are partly implemented by the inhibition of VSMC growth through ERK cascade. The hyperplasia of mammary gland epithelial cells could be significantly inhibited by raloxifene plus 17-betaE2.     

A non-calcemic Vitamin D analog modulates both nuclear and putative membranal estrogen receptors in cultured human vascular smooth muscle cells

In cultured human vascular smooth muscle cells (VSMC), estradiol-17beta (E2) induced a biphasic effect on DNA synthesis, i.e., stimulation at low concentrations and inhibition at high concentrations. Additionally, E2 increased the specific activity of creatine kinase (CK) in these cells. Observations that novel protein-bound membrane impermeant estrogenic complexes could elicit inhibition of DNA synthesis, suggested interaction via membranal binding sites. Nevertheless other effects, such as increasing CK activity were only seen with native E2 but not with E2-BSA, thus indicating that the classical nuclear receptor pathway was involved. In the present report, we confirm that human VSMC express both ERalpha and ERbeta. Further, pretreatment of cultured VSMC with the Vitamin D non-calcemic analog JK 1624 F2-2 (JKF) increased ERalpha mRNA (100-200%) but decreased ERbeta mRNA (30-40%) expression as measured by real time PCR. ERalpha protein expression assessed by Western blot analysis increased (25-50%) in parallel, whereas ERbeta protein expression declines (25-55%). Using ovalbumin bound to E2 (Ov-E2) linked to Eu (Eu-Ov-E2), to assess specific membrane binding sites, we observed that membranal binding was down regulated by JKF by 70-80%. In contrast, total cell binding of 3[H] E2, that nearly entirely represents intracellular E2 binding, was increased by 60-100% by the same Vitamin D analog. The results provide evidence that the effects of JKF on ERalpha/ERbeta as well as on membranal versus nuclear binding of estrogen are divergent and show differential modulation.     

The effects of peptides with estrogen‐like activity on cell proliferation and energy metabolism in human derived vascular smooth muscle cells

Hormone replacement therapy (HRT) for post‐menopausal symptoms in diabetes is associated with increased risk of coronary heart disease and stroke. Therefore, there is a need for new HRT with no adverse effects on diabetic post‐menopausal women. We developed peptides as potential estrogen mimetic compounds and now we evaluated the effects of the most efficacious peptide; hexapeptide estrogen‐mimetic peptide 1 (EMP‐1) (VSWFFE) in comparison to estrogen (E₂) and peptides with weak activity A44 (KAWFFE) and A45 (KRAFFE) on modulation of cell proliferation of vascular smooth muscle cells (VSMC) growing in normal (ng) or high glucose (hg) concentrations. In ng EMP‐1‐like E₂ inhibited cell proliferation at high concentration, and stimulated at low concentration. EMP‐1 did not affect E₂ stimulation of DNA, but inhibited E₂ inhibition of cell proliferation at high concentration. All effects by the combination of EMP‐1 and E₂ were abolished at hg. A44‐stimulated cell proliferation at all concentrations and A45 had no effect. When A44 was co‐incubated with E₂ at both concentrations, DNA synthesis was stimulated, but abolished at hg. A45 abolished E₂ stimulation and inhibition of cell proliferation at both glucose concentrations. All peptides tested except A45‐stimulated CK‐specific activity at both glucose concentrations. In hg A44 stimulation of DNA was unaffected as well as its inhibition by EMP‐1. EMP‐1 and A44 similar to E₂‐stimulated MAPK activity in ng or hg, suggesting similar mechanism of action. The results presented here suggest that EMP‐1 provided it acts similarly in vivo can replace E₂ for treatment of post‐menopausal women in hyperglycemia due to diabetes. J. Cell. Biochem. 110: 1142–1146, 2010. Published 2010 Wiley‐Liss, Inc.     

"Non-hypercalcemic" analogs of 1alpha,25 dihydroxy vitamin D augment the induction of creatine kinase B by estrogen and selective estrogen receptor modulators (SERMS) in osteoblast-like cells and rat skeletal organs

We have demonstrated previously that daily treatments for 3 days with the so-called "non-hypercalcemic" analogs of 1alpha,25 dihydroxy vitamin D in ROS 17/2.8 osteoblast-like cells, stimulate the specific activity of creatine kinase BB (CK), and that such treatment with these analogs followed by a single treatment with gonadal steroids, upregulates responsiveness and sensitivity to estradiol 17beta (E(2)) for the induction of CK. This study was designed to determine if these same "non-hypercalcemic" vitamin D analogs could upregulate in vivo the response to E(2) and whether substitution of selective estrogen receptor modulators (SERMS) for E(2) would result in the same upregulation. We found that one week or 2 weeks pretreatment of prepubertal rats with vitamin D analogs led to increased induction of CK by E(2) and by the SERMS tamoxifen, tamoxifen methiodide and raloxifene, in epiphysis and diaphysis of the femur but not in the uterus. However, in contrast to their antiestrogenic activity in the uterus, there was no inhibition of E(2) action by the SERMS in skeletal tissues. The induction of mRNA for ckb in ROS 17/2.8 cells by E(2) or SERMS was demonstrated only after vitamin D pretreatment; there was no inhibition of E(2) induction by SERMS. Antagonists of vitamin D dependent calcium transport (transcaltachia) did not inhibit stimulation by vitamin D analogs. These results support the involvement of a nuclear mechanism in the upregulation of induction of CK by E(2), which may be due, in part, to the ability of vitamin D to increase estrogen receptor(s).     

DT56a (Tofupill/Femarelle) selectively stimulates creatine kinase specific activity in skeletal tissues of rats but not in the uterus

The novel natural product DT56a (Tofupill/Femarelle), derived from soybean, has been shown to relieve menopausal vasomotor symptoms and to increase bone mineral density with no effect on sex steroid hormone levels or endometrial thickness. In the present study, we compared the effects of DT56a and estradiol-17beta (E2) on bone and cartilage (Ep) of immature or ovariectomized female rats, by measuring the changes in the specific activity of the BB isozyme of creatine kinase (CK). Single short-term injection of high doses of DT56a induced estrogenic activity in bones and uterus similar to that of E2. When administered in multiple oral doses, DT56a stimulated skeletal tissues similarly to E2, but whereas E2 increased CK specific activity in the uterus, DT56a did not. The selective estrogen receptor modulator (SERM) raloxifene (Ral) blocked the stimulation of CK by either DT56a or by E2 in all tissues tested. Our findings suggest that DT56a acts as a selective estrogen receptor modulator stimulating skeletal tissues without affecting the uterus. The effect of DT56a on other systems, such as the vascular and the central nervous system, are currently under investigation.     

Treatment with non-hypercalcemic analogs of 1,25-dihydroxyvitamin D3 increases responsiveness to 17beta-estradiol, dihydrotestosterone or raloxifene in primary human osteoblasts

Pretreatment with 1 nM 1,25-dihydroxyvitamin D(3) (1,25), or non-hypercalcemic Vitamin D analogs, upregulated the response of creatine kinase (CK) to 17beta-estradiol (30 nM E(2)), raloxifene (3000 nM RAL) or dihydrotestosterone (300 nM DHT) in primary human bone cells. Previously, we reported that these osteoblast-like cells responded to gonadal steroids in a sex specific manner. Bone cells derived from pre-menopausal women showed greater stimulation of CK specific activity by E(2) than bone cells from post-menopausal women; in male-derived cells no age related difference was found. In this study, we treated cells derived from female or male bones, at different ages, with the side chain modified analogs of Vitamin D: CB 1093 (CB), EB 1089 (EB), MC 1288 (MC) and the demonstrably non-calcemic hybrid analog JK 1624 F2-2 (JKF), by daily addition of 1 nM, for 3 days. On day 4, cells were incubated with sex steroids for 4h and cell extracts were prepared. Pretreatment with JKF or CB significantly upregulated the response to 30 nM E(2) in all female-derived cells and to 300 nM DHT in mature male-derived cells. In cells from older males, only JKF caused augmentation of DHT action. Bone cells from pre- or post-menopausal females responded to 3000 nM RAL by increased CK activity to the same extent as to 30 nM E(2); however, RAL and E(2), when applied together, resulted in mutual annihilation of their agonist activities. Vitamin D analogs prevented the antagonistic effect of RAL in the presence of E(2), possibly due to increased numbers of ERs. Both estrogen receptors, alpha (ERalpha) and beta (ERbeta), were expressed in male- as well as in female-derived cells. However, only in female-derived cells were ERalpha and ERbeta upregulated by pretreatment with Vitamin D analogs. This study raises the possibility of testing combined Vitamin D analog and estrogen replacement treatment for post-menopausal women to prevent osteoporosis.     

Estrogenic activity of glabridin and glabrene from licorice roots on human osteoblasts and prepubertal rat skeletal tissues

Data from both in vivo and in vitro experiments demonstrated that glabridin and glabrene are similar to estradiol-17β in their stimulation of the specific activity of creatine kinase, although at higher concentrations, but differ in their extent of action and interaction with other drugs. In pre-menopausal human bone cells, the response to estradiol-17β and glabridin (at higher concentration) was higher than in post-menopausal cells; whereas, glabrene (at higher concentration) was more effective in post-menopausal cells. At both ages, the response to estradiol-17β and glabridin was enhanced by pretreatment with the less-calcemic Vitamin D analog CB 1093 (CB) and the demonstrably non-calcemic analog JK 1624 F₂-2 (JKF). The response to glabrene was reduced by this pretreatment. Both glabridin and glabrene stimulated creatine kinase specific activity in diaphyseal bone and epiphyseal cartilage of prepubertal female rats. Daily feeding (3–14 days) of prepubertal female rats with glabridin, estradiol-17β or their combination, also stimulated creatine kinase specific activity. Glabridine, similarly to estradiol-17β, also stimulated creatine kinase specific activity in ovariectomized female rats. Raloxifene, in combination with glabridin or estradiol-17β, demonstrated the phenomenon of mutual annihilation of stimulation of creatine kinase specific activity in both epiphysis and diaphysis. Glabrene activity was not inhibited by raloxifene. Therefore, glabridin shows greater similarity to estradiol-17β and thus greater potential, with or without Vitamin D, to modulate bone disorders in post-menopausal women.     

Modulation of the response to estradiol-17beta of rat vascular tissues by a non calcemic vitamin D analog

Estradiol-17beta (E(2)) increases creatine kinase (CK) specific activity in aorta (Ao) and left ventricle of the heart (Lv) from rat females. In the present study, we analyzed the effects of pretreatment with the non calcemic analog of vitamin D, JK 1624 F2-2 (JKF) on the response to E(2) (either 0.5 or 5 microg/rat) of Ao and Lv from prepubertal female rats. JKF did not affect CK in either organ. However, pretreatment with JKF (0.1 ng/g body weight for 1 or 2 weeks) increased the CK response to E(2) (0.5 microg/rat) by 50 +/- 10% in Ao and by 150 +/- 12% in Lv. The CK response to 5 microg/rat of E(2) in intact female rats, was increased by 118 +/- 15% and 99 +/- 11% in the Ao and by 89 +/- 6% and 112 +/- 13% in the Lv, in animals treated daily with JKF for 1 or 2 weeks, respectively, before administration of E(2). JKF also increased the response to 500 microg/rat raloxifene (Ral) by 47 +/- 8% in Ao and by 56 +/- 12% in Lv. Preliminary experiments showed that JKF treatment induced a approximately 50% increase in estradiol receptor ERalpha in both organs. The results indicate that the vitamin D analog JKF upregulates the response and sensitivity of vascular tissues to E(2), in association with increased expression of their ERalpha. These results should prompt examination of the possibility that the effects estrogen therapy in postmenopausal women can be augmented by vitamin D or its analogs.     

DT56a (Femarelle): a natural selective estrogen receptor modulator (SERM)

A selective estrogen receptor modulator (SERM) is defined as a substance with dissimilar effects on different tissues: agonist in some and antagonists in others. The natural compound DT56a (Femarelle) was shown to activate estrogen receptors in human cultured female derived osteoblasts. It was also shown to relieve menopausal symptoms and to increase bone mineral density with no effect on sex steroid hormone levels and on the endometrial thickness. DT56a, similarly to estradiol-17β (E₂), stimulated the specific activity of creatine kinase (CK) in skeletal and vascular tissues of female rats, as a marker of estrogen receptor (ER) activation. However, in the uterus, CK was activated only by E₂ but not by DT56a. In order to prove that DT56a is a SERM, we examined the mutual interaction between DT56a and E₂, at supra physiological doses, in different tissues in both intact and ovariectomized female rats, as well as in human cultured vascular and bone cells. Administration of DT56a or E₂ stimulated CK in all tissues tested, but when given simultaneously, in intact immature female rats, DT56a completely abolished E₂ stimulation of CK in all organs except in the diaphyseal bone where the inhibition was partial. In ovariectomized female rats, DT56a abolished E₂'s stimulation of CK in diaphyseal bone, thymus, uterus and pituitary but caused a partial inhibition in aorta, left ventricle and epiphyseal cartilage. In human bone cells E₂ stimulation of CK, of alkaline phosphatase (AP) activity and of DNA synthesis was completely abolished by DT56a in post-menopausal cells and partially inhibited in pre-menopausal cells. In human vascular cells, inhibition of DNA synthesis by E₂ was completely abolished by DT56a and E₂-induced CK was partially inhibited by DT56a. The results support the finding that DT56a is a SERM; it stimulated different parameters similar to E₂, but when given simultaneously, at supra physiological doses, inhibited these E₂'s effects. Further investigations regarding intra and extra cellular mechanism of action of DT56a are currently performed.     

Comparative study of the short-term effects of a novel selective estrogen receptor modulator, ospemifene, and raloxifene and tamoxifen on rat uterus

To investigate the differential short-term effects of selective estrogen receptor (ER) modulators (SERMs) on uterus, we treated adult ovariectomized rats with a novel SERM, ospemifene (Osp), two previously established SERMs (tamoxifen and raloxifene (Ral)) and estradiol. The expression of two estrogen-regulated early response genes c-fos and vascular endothelial growth factor (VEGF), and DNA synthesis were analysed at 1-24 h after treatment of ovariectomized rats. Induction of c-fos mRNA by each of the SERMs showed a biphasic pattern with peaks at 3 and 20 h, respectively. The maximum level of VEGF mRNA was observed at 1 h after raloxifene and 6 h after tamoxifen or ospemifene treatment. Maximum levels of the c-fos and VEGF mRNA after raloxifene treatment were higher than those seen after treatments with E2 or a corresponding dose of tamoxifen or ospemifene. DNA synthesis was significantly increased by ospemifene, tamoxifen and raloxifene both in luminal and glandular epithelium. The stimulation was transient, peaking at 16 h. In comparison, the maximum level observed at 16 h after E2 treatment sustained at least until 24 h. DNA synthesis in stromal cells was increased by the SERMs but not by E2 at 24 h. When treated together with E2, the SERMs were able to antagonise E2-stimulated DNA synthesis at 16 h. Our results demonstrate that the initial response of uterus to ospemifene, raloxifene and tamoxifen includes activation of early response genes and even transient stimulation of DNA synthesis in spite of their different long-term effects. However, the early stimulatory events may be mediated by different mechanisms leading to diverging pathways in various tissue compartments and development of differential SERM-specific long-term responses of uterus.     

Estrogen-like activity of glabrene and other constituents isolated from licorice root

Licorice root extract and its major isoflavan, glabridin, exhibited varying degrees of estrogen receptor (ER) agonism in different tissues in vitro and in vivo. Animals fed with licorice extract, compared with estradiol and glabridin, showed an increase in creatine kinase (CK) activity, a known marker for estrogen responsive genes, which was higher than expected from the levels of glabridin in the extract. This led us to test for other components that may contribute to this strong estrogen agonist activity. Results indicated that glabrene and isoliquiritigenin, (2',4',4-three hydroxy chalcone) (ILC) in the licorice extract can bind to the human ER with higher affinity (IC50, 1 and 0.5 microM) than glabridin (IC50, 5 microM). The stimulatory effects of glabrene in vivo were tissue specific and similar to those of estradiol. The effect of increasing concentrations of glabrene and ILC on the growth of breast tumor cell were biphasic. Both showed an ER-dependent growth-promoting effect at low concentrations (10 nM-10 microM), and ER-independent antiproliferative activity at concentrations >15 microM. This is the first study to indicate that glabrene, an isoflavene exerted varying degrees of ER agonism in different tissues.