Neonatal agonism of ERβ impairs male reproductive behavior and attractiveness

The organization of the developing male rodent brain is profoundly influenced by endogenous steroids, most notably estrogen. This process may be disrupted by estrogenic endocrine disrupting compounds (EDCs) resulting in altered sex behavior and the capacity to attract a mate in adulthood. To better understand the relative role each estrogen receptor (ER) subtype (ERα and ERβ) plays in mediating these effects, we exposed male Long Evans rats to estradiol benzoate (EB, 10 μg), vehicle, or agonists specific for ERβ (DPN, 1 mg/kg) or ERα (PPT, 1 mg/kg) daily for the first four days of life, and then assessed adult male reproductive behavior and attractiveness via a partner preference paradigm. DPN had a greater adverse impact than PPT on reproductive behavior, suggesting a functional role for ERβ in the organization of these male-specific behaviors. Therefore the impact of neonatal ERβ agonism was further investigated by repeating the experiment using vehicle, EB and additional DPN doses (0.5 mg/kg, 1 mg/kg, and 2 mg/kg bw). Exposure to DPN suppressed male reproductive behavior and attractiveness in a dose dependent manner. Finally, males were exposed to EB or an environmentally relevant dose of genistein (GEN, 10 mg/kg), a naturally occurring xenoestrogen, which has a higher relative binding affinity for ERβ than ERα. Sexual performance was impaired by GEN but not attractiveness. In addition to suppressing reproductive behavior and attractiveness, EB exposure significantly lowered the testis to body weight ratio, and circulating testosterone levels. DPN and GEN exposure only impaired behavior, suggesting that disrupted androgen secretion does not underlie the impairment.     

Influence of ERβ selective agonism during the neonatal period on the sexual differentiation of the rat hypothalamic-pituitary-gonadal (HPG) axis

Background

It is well established that sexual differentiation of the rodent hypothalamic-pituitary-gonadal (HPG) axis is principally orchestrated by estrogen during the perinatal period. Here we sought to better characterize the mechanistic role the beta form of the estrogen receptor (ERβ) plays in this process.

Methods

To achieve this, we exposed neonatal female rats to three doses (0.5, 1 and 2 mg/kg) of the ERβ selective agonist diarylpropionitrile (DPN) using estradiol benzoate (EB) as a positive control. Measures included day of vaginal opening, estrous cycle quality, GnRH and Fos co-localization following ovariectomy and hormone priming, circulating luteinizing hormone (LH) levels and quantification of hypothalamic kisspeptin immunoreactivity. A second set of females was then neonatally exposed to DPN, the ERα agonist propyl-pyrazole-triol (PPT), DPN+PPT, or EB to compare the impact of ERα and ERβ selective agonism on kisspeptin gene expression in pre- and post-pubescent females.

Results

All three DPN doses significantly advanced the day of vaginal opening and induced premature anestrus. GnRH and Fos co-labeling, a marker of GnRH activation, following ovariectomy and hormone priming was reduced by approximately half at all doses; the magnitude of which was not as large as with EB or what we have previously observed with the ERα agonist PPT. LH levels were also correspondingly lower, compared to control females. No impact of DPN was observed on the density of kisspeptin immunoreactive (-ir) fibers or cell bodies in the arcuate (ARC) nucleus, and kisspeptin-ir was only significantly reduced by the middle (1 mg/kg) DPN dose in the preoptic region. The second experiment revealed that EB, PPT and the combination of DPN+PPT significantly abrogated preoptic Kiss1 expression at both ages but ARC expression was only reduced by EB.

Conclusion

Our results indicate that selective agonism of ERβ is not sufficient to completely achieve male-typical HPG organization observed with EB or an ERα agonist.     

Impact of estrogen receptor alpha and beta agonists on delayed alternation in middle-aged rats

Estrogens act in the adult brain to modulate cognition, enhancing performance on some learning tests and impairing performance on others. Our previous research has revealed an impairing effect of chronic 17β-estradiol treatment in young and aged rats on a prefrontally-mediated working memory task, delayed spatial alternation (DSA). Little is known about the mechanisms of these impairing effects. The current study examined the effects of selective estrogen receptor (ER) α or ERβ activation on DSA performance in middle-aged female rats. Ovariectomized 12 month old Long–Evans (LE) rats were treated by subcutaneous injection with the ERα agonist propyl pyrazole triol (PPT) or the ERβ agonist diarylpropionitrile (DPN) at 0.02, 0.08, or 0.20 mg/kg/day, or with oil vehicle and tested on an operant variable delay DSA task. A 17β-estradiol group (10% in cholesterol) was included as a positive control group. We replicated our previous finding of a 17β-estradiol induced deficit on DSA performance and this effect was paralleled by low dose (0.02 mg/kg/day) DPN treatment. Higher doses of DPN failed to produce a significant change in performance. The highest dose of PPT (0.20 mg/kg/day) also impaired performance, but this effect was subtle and limited to the longest delay during the final block of testing. These data confirm our earlier findings that chronic 17β-estradiol treatment has an impairing effect on the DSA task, and suggest that ERβ activation may underlie the deficit.     

Impact of estrogen receptor alpha and beta agonists on delayed alternation in middle-aged rats

Estrogens act in the adult brain to modulate cognition, enhancing performance on some learning tests and impairing performance on others. Our previous research has revealed an impairing effect of chronic 17β-estradiol treatment in young and aged rats on a prefrontally-mediated working memory task, delayed spatial alternation (DSA). Little is known about the mechanisms of these impairing effects. The current study examined the effects of selective estrogen receptor (ER) α or ERβ activation on DSA performance in middle-aged female rats. Ovariectomized 12 month old Long–Evans (LE) rats were treated by subcutaneous injection with the ERα agonist propyl pyrazole triol (PPT) or the ERβ agonist diarylpropionitrile (DPN) at 0.02, 0.08, or 0.20 mg/kg/day, or with oil vehicle and tested on an operant variable delay DSA task. A 17β-estradiol group (10% in cholesterol) was included as a positive control group. We replicated our previous finding of a 17β-estradiol induced deficit on DSA performance and this effect was paralleled by low dose (0.02 mg/kg/day) DPN treatment. Higher doses of DPN failed to produce a significant change in performance. The highest dose of PPT (0.20 mg/kg/day) also impaired performance, but this effect was subtle and limited to the longest delay during the final block of testing. These data confirm our earlier findings that chronic 17β-estradiol treatment has an impairing effect on the DSA task, and suggest that ERβ activation may underlie the deficit.     

Synthesis and structure–activity relationships of 1-benzylindane derivatives as selective agonists for estrogen receptor beta

The estrogen receptor beta (ERβ) selective agonist is considered a promising candidate for the treatment of estrogen deficiency symptoms in ERβ-expressing tissues, without the risk of breast cancer, and multiple classes of compounds have been reported as ERβ selective agonists. Among them, 6-6 bicyclic ring-containing structures (e.g., isoflavone phytoestrogens) are regarded as one of the cyclized analogues of isobutestrol 5b, and suggest that other cyclized scaffolds comprising 5-6 bicyclic rings could also act as selective ERβ ligands. In this study, we evaluated the selective ERβ agonistic activity of 1-(4-hydroxybenzyl)indan-5-ol 7a and studied structure–activity relationship (SAR) of its derivatives. Some functional groups improved the properties of 7a; introduction of a nitrile group on the indane-1-position resulted in higher selectivity for ERβ (12a), and further substitution with a fluoro or a methyl group to the pendant phenyl ring was also preferable (12b, d, and e). Subsequent chiral resolution of 12a identified that R-12a has a superior profile over S-12a. This is comparable to diarylpropionitrile (DPN) 5c, one of the promising selective ERβ agonists and indicates that this indane-based scaffold has the potential to provide better ERβ agonistic probes.     

Reproductive experience modifies the effects of estrogen receptor alpha activity on anxiety-like behavior and corticotropin releasing hormone mRNA expression

Previous studies have demonstrated that prior reproductive experience can influence anxiety-like behaviors, although neural mechanisms underlying this shift remain unknown. Studies in virgin females suggest that activation of the two estrogen receptor subtypes, ERα and ERβ, have differing effects on anxiety. Specifically, ERβ activation has been shown to reduce anxiety-like behaviors, while ERα activation has no significant effect. The purpose of the present study was to examine the possible roles of ERα and ERβ subtypes in parity-induced alterations in anxiety-like behavior, as tested on the elevated plus maze (EPM). Groups of ovariectomized, age-matched, nulliparous and primiparous females were tested on the EPM following administration of the ERα agonist 4,4',4'-(4-Propyl-{1H}-pyrazole-1,3,5-tryl)trisphenol (PPT; 1 mg/kg), the ERβ agonist Diarylpropionitrile (DPN; 1 mg/kg) or vehicle (DMSO). All drugs were administered once daily for 4 days prior to testing as this dosing paradigm has previously been used to demonstrate anxiolytic effects of DPN in virgin rats. In addition, as exposure to the EPM is a psychological stressor, physiological markers of the stress response were measured in both plasma (corticosterone) and brain (corticotropin releasing hormone; CRH) post-EPM testing. Unexpectedly, the ERα agonist PPT selectively increased the time spent exploring the open arms of the EPM in non-lactating, primiparous females, with no significant effects of DPN observed in either nulliparous or primiparous subjects. All females administered PPT and tested on the EPM demonstrated significantly reduced corticosterone secretion when compared to vehicle-treated controls. In addition, significant effects of both reproductive experience and PPT administration on CRH mRNA expression were observed in both the paraventricular nucleus and amygdala using qPCR. These findings indicate that reproductive experience modulates the effects of ERα activation on both EPM behavior related to anxiety and CRH gene expression.     

Estrogen receptor-β activation in combination with letrozole blocks the growth of breast cancer tumors resistant to letrozole therapy

Treatment with anti-estrogens or aromatase inhibitors (AI) is the main therapeutic strategy used against estrogen receptor ERα-positive breast cancer. Resistance to these therapies presents a major challenge in the management of breast cancer. Little is known about ERβ in breast carcinogenesis. Our aim in this study is to examine potential novel strategies utilizing ERβ activity to overcome AI resistance. We provide evidence that ERβ agonist can reduce the growth of AI-resistant breast cancer cells. Our data further confirm that therapeutic activation of ERβ by DPN, an ERβ agonist, blocks letrozole-resistant tumor growth in a xenograft model. Interestingly, DPN exerted tumor growth inhibition only in the presence of the AI letrozole, suggesting that combination therapy including ERβ activators and AI may be used in the clinical setting treating AI resistant breast cancer. An increase in ERβ levels, with diminished ERα/ERβ ratio, was observed in the tumors from mice treated with DPN/letrozole combination compared to single agents and control. Decreased Cyclin D1 and increased CyclinD1/CDK inhibitors p21 and p27 levels in DPN/letrozole treated tumors were observed, suggesting that the combination treatment may inhibit tumor growth by blocking G1/S phase cell cycle progression. Our data show a decrease in MAPK phosphorylation levels without affecting total levels. In addition to providing evidence suggesting the potential use of ERβ agonists in combination with letrozole in treating AI resistant breast cancer and prolonging sensitivity to AI, we also provide mechanistic evidence supporting the role of ERβ in altering the expression profile associated with resistance.     

Sex specific response of cultured human bone cells to ERα and ERβ specific agonists by modulation of cell proliferation and creatine kinase specific activity

We have previously reported that human cultured bone cells (hObs) respond to estradiol-17β (E2) by stimulating DNA synthesis, creatine kinase BB specific activity (CK) and other parameters sex-specifically. We now investigate the sex specificity of the response of these hObs to estrogen receptor (ER) α and ERβ specific agonists. Real time PCR revealed that all cells express mRNA for both ERs. ERα mRNA but not ERβ mRNA was stimulated by all estrogenic compounds in both pre- and post-menopausal hObs with no effect in male hObs. Cells treated with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ specific agonist) and 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα specific agonist) showed increased DNA synthesis and CK in all female but not male hObs. Raloxifene (Ral), a specific ERα antagonist, inhibited the stimulation of DNA synthesis and CK by E2 or PPT, but not by DPN. DPN and PPT like E2 modulated the expression of both 12 and 15 lipooxygenase (LO) mRNA in both female but not male hObs. 12 and 15 HETE production was modulated only by DPN and PPT in these cells. The LO inhibitor baicaleine inhibited only E2 and PPT but not DPN effects in both female hObs. In conclusion, we provide herein evidence for the separation of age- and sex-dependent mediation via both ERα and ERβ pathways in the effects of estrogens on hObs, with a yet unknown mechanism.     

Estrogen rescues heart failure through estrogen receptor Beta activation

Background

Recently, we showed that exogenous treatment with estrogen (E2) rescues pre-existing advanced heart failure (HF) in mice. Since most of the biological actions of E2 are mediated through the classical estrogen receptors alpha (ERα) and/or beta (ERβ), and both these receptors are present in the heart, we examined the role of ERα and ERβ in the rescue action of E2 against HF.

Methods

Severe HF was induced in male mice by transverse aortic constriction-induced pressure overload. Once the ejection fraction (EF) reached ~?35%, mice were treated with selective agonists for ERα (PPT, 850 μg/kg/day), ERβ (DPN, 850 μg/kg/day), or E2 (30 μg/kg/day) together with an ERβ-antagonist (PHTPP, 850 μg/kg/day) for 10 days.

Results

EF of HF mice was significantly improved to 45.3?±?2.1% with diarylpropionitrile (DPN) treatment, but not with PPT (31.1?±?2.3%). E2 failed to rescue HF in the presence of PHTPP, as there was no significant improvement in the EF at the end of the 10-day treatment (32.5?±?5.2%). The improvement of heart function in HF mice treated with ERβ agonist DPN was also associated with reduced cardiac fibrosis and increased cardiac angiogenesis, while the ERα agonist PPT had no significant effect on either cardiac fibrosis or angiogenesis. Furthermore, DPN improved hemodynamic parameters in HF mice, whereas PPT had no significant effect.

Conclusions

E2 treatment rescues pre-existing severe HF mainly through ERβ. Rescue of HF by ERβ activation is also associated with stimulation of cardiac angiogenesis, suppression of fibrosis, and restoration of hemodynamic parameters.

     

Chronic treatment with estrogen receptor agonists restores acquisition of a spatial learning task in young ovariectomized rats

Previous work has shown that continuous estradiol replacement in young ovariectomized rats enhances acquisition of a delayed matching-to-position (DMP) T-maze task over that of ovariectomized controls. The mechanism by which estradiol confers this benefit has not been fully elucidated. This study examined the role of selective estrogen receptor agonists of ERα, ERβ, and GPR30 in the enhancement of spatial learning on a DMP task by comparing continuous estradiol replacement with continuous administration of PPT (an agonist of ERα), DPN (an agonist of ERβ), or G-1 (an agonist of GPR30) relative to gonadally intact and ovariectomized vehicle-treated controls. It was found that ovariectomy impaired acquisition on this task, whereas all ER selective agonists restored the rate of acquisition to that of gonadally intact controls. These data suggest that estradiol can work through any of several estrogen receptors to enhance the rate of acquisition on this task.     

Enhanced estrogen receptor beta (ERβ) selectivity of fluorinated carborane-containing ER modulators

We previously showed that fluorination of the carborane-containing selective estrogen receptor modulator (SERM) BE360 altered the agonist/antagonist activity balance and the estrogen receptor (ER) α/β subtype selectivity. Here, we designed and synthesized a series of fluorinated carboranyl phenols as candidate ERβ-selective ligands. Introduction of a fluorine atom onto the carborane cage commonly reduced the binding affinity for ERα, to an extent that depended on the other substituents present. The B-fluorinated m-carboranyl phenol 4a showed fourfold more potent ERβ-binding affinity than the parent non-fluorinated compound 7. 1-Iodo-9-fluoro-m-carboranyl phenol 4f showed high ERβ-binding affinity with an ERβ/ERα selectivity ratio of 8.2. Among the compounds tested, 6 showed the highest ERβ selectivity (10.1-fold) and the highest ER-agonistic activity (EC₅₀: 5.1 × 10^?10 M) in MCF-7 cell proliferation assay.     

Effects of postnatal estrogen manipulations on juvenile alloparental behavior

Sex- and species-specific patterns of estrogen receptor (ER)-α expression are established early in development, which may contribute to sexual differentiation of behavior and determine male social organization. The current study investigated the effects of ERα and ERβ activation during the second postnatal week on subsequent alloparental behavior and ERα expression in juvenile prairie voles. Male and female pups were treated daily with 17β-estradiol (E2, ERα/ERβ agonist), PPT (selective ERα agonist), DPN (selective ERβ agonist), or the oil vehicle on postnatal days (PD) 8–14. Alloparental behavior and ERα expression were examined at PD21. PPT treatment inhibited prosocial motivation in males and increased pup-directed aggression in both sexes. E2 and DPN had no apparent effect on behavior in either sex. PPT-treated males had increased ERα expression in the medial preoptic area (MPN), medial amygdala (MEApd) and bed nucleus of the stria terminalis (BSTpr). DPN treatment also increased ERα expression in males, but only in the BSTpr. Female ERα expression was unaffected by treatment. These results support the hypothesis that ERα activation in early life is associated with less prosocial patterns of central ERα expression and alloparental behavior in males. The lack of an effect of E2 on behavior suggests that ERβ may antagonize the effects of ERα on alloparental behavior. The results in DPN-treated males suggest that ERα in the MEApd, and not the BSTpr, may be a primary determinant of alloparental behavior in males.     

The effects of estrogen receptors α- and β-specific agonists and antagonists on cell proliferation and energy metabolism in human bone cell line

In cultured human osteoblasts estradiol-17β (E2) modulated DNA synthesis, the specific activity of creatine kinase BB (CK), 12 and 15 lipoxygenase (LO) mRNA expression and formation of 12- and 15-hydroxyeicosatetraenoic acid (HETE). We now investigate the response of human bone cell line (SaOS2) to phytoestrogens and estrogen receptors (ER)-specific agonists and antagonists. Treatment of SaSO2 with E2, 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; ERβ-specific agonist), 4,4',4″-[4-propyl-(1H)-pyrazol-1,3,5-triyl] tris-phenol (PPT; ERα-specific agonist), biochainin A (BA), daidzein (D), genistein (G) and raloxifene (Ral) showed increased DNA synthesis and CK. Ral inhibited completely all stimulations except DPN and to some extent D. The ERα-specific antagonist methyl-piperidino-pyrazole (MPP) and the ERβ-specific antagonist 4-[2-phenyl-5,7-bis (tri-fluoro-methyl) pyrazolo [1,5-a]pyrimidin-3-yl] phenol (PTHPP) inhibited DNA synthesis, CK and reactive oxygen species (ROS) formation induced by estrogens according to their receptors affinity. The LO inhibitor baicaleine inhibited only E2, DPN and G's effects. E2 and Ral unlike all other compounds had no effect on ERα mRNA expression, while ERβ mRNA expression was stimulated by all compounds. All compounds modulated the expression of 12LO and 15LO mRNA, except E2, PPT and Ral for 12LO, and 12- and 15-HETE productions and stimulated ROS formation which was inhibited by NADPH oxidase inhibitors diphenyleneiodonium chloride (DPI) and N-acetyl cysteine and the estrogen inhibitor ICI. DPI did not affect hormonal-induced DNA and CK. In conclusion, we provide evidence for the separation of mediation via ERα and ERβ pathways in the effects of estrogenic compounds on osteoblasts, but the role of LO/HETE/ROS is unclear.     

雌激素和多巴胺对大鼠垂体组织中雌激素受体基因表达的影响

目的研究雌激素和多巴胺激动剂对雌激素受体在大鼠垂体组织表达的作用。方法20只成年雌性Wistar大鼠,切除卵巢后,随机分2组：（1）对照组（n=5）,皮下植入空白硅胶管;（2）雌激素组（n=15）皮下植入含有乙烯雌酚的硅胶管,8周后,两组各处死5只大鼠,雌激素组剩余大鼠（n=10）取出硅胶管,随机再分2组,安慰剂组（n=5）给予自来水灌胃,多巴胺组（n=5）给予溴隐亭（多巴胺激动剂）灌胃,用药4周后处死动物。放免法测定血清PRL水平,用反转录一聚合酶链反应（RT-PCR）方法检测ERs在各组垂体组织中的表达,以β-actin作为内参照,借助于计算机凝胶成像系统分析表达量。结果ERa,ERβ以及TERP在各组大鼠垂体组织均有表达,其中ERα和TERPmRNA水平在雌激素组明显高于对照组（P〈0．001）,在安慰剂组和多巴胺组的表达无明显差别。结论大鼠垂体组织中存在ER的表达,雌激素对ERα和TERP的表达具有升调节作用,多巴胺不影响雌激素受体的表达。     

Neonatal exposure to endocrine active compounds or an ERbeta agonist increases adult anxiety and aggression in gonadally intact male rats

Endocrine active compounds (EACs) have been shown to influence a number of reproductive endpoints but less is known about how they might affect other hormone dependent behaviors including anxiety and aggression. Recent evidence suggests that these effects may be mediated through the beta form of the estrogen receptor (ERbeta). Using male Long Evans rats, we sought to determine how neonatal exposure to EACs affects anxiety and aggression in adulthood. Anxiety was assessed using the elevated plus maze and aggression was assessed 8 weeks later using the resident intruder test. To gain insight into which ER subtype (ERalpha vs ERbeta) might be mediating these effects we used agonists specific for ERalpha (1,3,5-tris(4-Hydroxyphenyl)-4-propyl-1H-pyrazole (PPT)) or ERbeta (Diarylpropionitrile (DPN)) as additional treatment groups. For these experiments the synthetic EAC bisphenol-A (BPA) and the phytoestrogen metabolite equol (EQ) were used. Male neonates were injected with either 0.05 ml sesame oil (control), 50 microg estradiol benzoate (EB), 1 mg/kg DPN, 1 mg/kg PPT, 50 microg/kg BPA, or 10 mg/kg EQ daily for 4 days beginning on the day of birth (PND 0). Compared to the oil treated controls, significantly fewer open arm entries were made by the males neonatally treated with DPN, EQ, or BPA. The DPN and EQ treated males were also more aggressive compared to the controls. These findings suggest that neonatal exposure to EACs with agonistic activity on ERbeta may influence affective behavior in adulthood, including anxiety and aggression.     

Psoralidin,a coumestan analogue,as a novel potent estrogen receptor signaling molecule isolated from Psoralea corylifolia

A novel biological activity of psoralidin as an agonist for both estrogen receptor (ER)α and ERβ agonist has been demonstrated in our study. Psoralidin has been characterized as a full ER agonist, which activates the classical ER-signaling pathway in both ER-positive human breast and endometrial cell lines as well as non-human cultured cells transiently expressing either ERα or ERβ. The estrogenic activity was determined using the relative expression levels of either reporter or the endogenous genes dependent on the agonist-bound ER to the estrogen response element (ERE). Psoralidin at 10 μM was able to induce the maximum reporter gene expression corresponding to that of E₂-treated cells and such activation of the ERE-reporter gene by psoralidin was completely abolished by the cotreatment of a pure ER antagonist, implying that the biological activities of psoralidin are mediated by ER. Psoralidin was also able to induce the endogenous estrogen-responsive gene, pS2, in human breast cancer cells MCF-7. It was observed that activation of the classical ER-signaling pathway by psoralidin is mediated via induction of ER conformation by psoralidin and direct binding of the psoralidin–ER complex to the EREs present in the promoter region of estrogen-responsive genes, as shown by chromatin immunoprecipitation assay results. Finally, molecular docking of psoralidin to the ligand binding pocket of the ERα showed that psoralidin is able to mimic the binding interactions of E₂, and thus, it could act as an ER agonist in the cellular environment.     

Anti-proliferative effects of a novel isoflavone derivative in medullary thyroid carcinoma: An in vitro study

Currently available treatments for patients with medullary thyroid carcinoma (MTC) with residual or recurrent disease after primary surgery have low efficacy rates. In view of the possible role of estrogen in the development of thyroid neoplasia, we explored whether proliferation of the human MTC TT cell line, might be curbed by carboxy-daidzein-tBoc (cD-tBoc), a novel isoflavone derivative. Estrogen receptor (ER) α mRNA expression in TT cells was more abundant than ERβ, with a ratio of 48:1. Estradiol-17β (E2) increased DNA synthesis in a dose dependent manner. [(3)H]-thymidine incorporation was also stimulated by the ERβ agonist DPN and the ERα agonist PPT. cD-tBoc inhibited TT cell growth as assessed by thymidine incorporation, XTT assay, and microscopic analysis of culture wells. Creatine kinase specific activity, a marker of the modulatory effects of estrogen on cell energy metabolism, was likewise inhibited. The inhibitory effect of cD-tBoc on [(3)H]-thymidine incorporation could be blocked by the ERβ antagonist PTHPP but not by the ERα antagonist MPP, suggesting that the antiproliferative effect of cD-tBoc on these cells is mediated through ERβ. Furthermore, cD-tBoc potently increased apoptosis and cell necrosis. Co-incubation with the antiapoptotic agent Z-VAD-FMK reversed the growth inhibitory effect elicited by cD-tBoc. These results support the hypothesis that estrogens are involved in the proliferation of MTC. The potent anti-proliferative effects mediated by isoflavone derivatives in the human MTC cell line TT suggest and that this property may be utilized to design effective anti-neoplastic agents.