Estrogen receptor-alpha mediates the effects of estradiol on telomerase activity in human mesenchymal stem cells

Sex steroid hormone receptors play a central role in modulating telomerase activity, especially in cancer cells. However, information on the regulation of steroid hormone receptors and their distinct functions on telomerase activity within the mesenchymal stem cell are largely unavailable due to low telomerase activity in the cell. In this study, the effects of estrogen (E2) treatment and function of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) on telomerase activity were investigated in human mesenchymal stem cells (hMSCs). Telomerase activity and mRNA expression of the catalytic subunit of telomerase (hTERT) were upregulated by treatment of the cells with E2. The protein concentration of ERalpha was also increased by E2 treatment, and enhancement of ERalpha accumulation in the nucleus was clearly detected with immunocytochemistry. When ERalpha expression was reduced by siRNA transfection into hMSCs, the effect of E2 on the induction of hTERT expression and telomerase activity was diminished. In contrast, the transient overexpression of ERalpha increased the effect of E2 on the expression of hTERT mRNA. These findings indicate that the activation of hTERT expression and telomerase activity by E2 in hMSCs depends on ERalpha, but not on ERbeta.     

17Beta-estradiol triggers postspawning in spermatogenically active gilthead seabream (Sparus aurata L.) males

The testis is a tightly controlled dynamic tissue. In mammals, there is growing evidence that estrogen plays a role in the regulation of testicular functions. In teleosts, high levels of 17beta-estradiol (E2) in serum correlate with the end of spermatogenesis, spawning, and the initiation of postspawning stages when spermatogonia are the main cell types in the testis. Moreover, E2 modulates leukocyte functions in several teleost species. We hypothesized, therefore, that E2 would induce the infiltration of acidophilic granulocytes and cause a resumption of testicular cell proliferation in spermatogenically active gilthead seabream males. Several studies of this species have reported that supraphysiological doses of E2 are needed to induce histological and developmental changes in males. In fact, as gilthead seabream is a protandrous hermaphrodite teleost, long exposures (6-14 wk) to high doses of E2 result in feminization of the males. Taking all this into account, we sharply increased E2 levels during short times by i.p. injecting E2 diluted in coconut oil as the vehicle and sampled the fish after 7, 13, and 18 days to assess the effects that E2 had on spermatogenesis. It was observed that E2 levels in plasma increased, while 11-ketotestosterone (11-KT) and testosterone (T) levels remained unaltered. However, 11-KT and T levels strongly increased in control fish 18 days postinjection. The most relevant result of our study was that E2 accelerates the final events of spermatogenesis, inhibits the proliferation of spermatogonia in early stages, and induces some of the processes that usually occur during postspawning, such as the infiltration of acidophilic granulocytes and the apoptosis of primary spermatogonia. Strikingly, neither the shedding of spermatozoa nor an increase in the proliferative rate of spermatogonia stem cells was observed, probably because of the lack of other necessary stimuli, such as the increase in T levels that takes place during normal postspawning.     

Rapid actions of plasma membrane estrogen receptors regulate motility of mouse embryonic stem cells through a profilin-1/cofilin-1-directed kinase signaling pathway

Long-term estrogen actions are vital for driving cell growth, but more recent evidence suggests that estrogen mediates more rapid cellular effects. However, the function of estradiol-17β (E(2))-BSA in mouse embryonic stem cells has not been reported. Therefore, we examined the role of E(2)-BSA in mouse embryonic stem cell motility and its related signal pathways. E(2)-BSA (10(-8) m) significantly increased motility after 24 h incubation and increased filamentous (F)-actin expression; these effects were inhibited by the estrogen receptor antagonist ICI 182,780, indicating that E(2)-BSA bound membrane estrogen receptors and initiated a signal. E(2)-BSA increased c-Src and focal adhesion kinase (FAK) phosphorylation, which was attenuated by ICI 182,780. The E(2)-BSA-induced increase in epidermal growth factor receptor (EGFR) phosphorylation was inhibited by Src inhibitor PP2. As a downstream signal molecule, E(2)-BSA activated cdc42 and increased formation of a complex with the neural Wiskott-Aldrich syndrome protein (N-WASP)/cdc42/transducer of cdc42-dependent actin assembly-1 (TOCA-1), which was inhibited by FAK small interfering RNA (siRNA) and EGFR inhibitor AG 1478. In addition, E(2)-BSA increased profilin-1 expression and cofilin-1 phosphorylation, which was blocked by cdc42 siRNA. Subsequently, E(2)-BSA induced an increase in F-actin expression, and cell motility was inhibited by each signal pathway-related siRNA molecule or inhibitors but not by cofilin-1 siRNA. A combined treatment of cofilin-1 siRNA and E(2)-BSA increased F-actin expression and cell motility more than that of E(2)-BSA alone. These data demonstrate that E(2)-BSA stimulated motility by interacting with profilin-1/cofilin-1 and F-actin through FAK- and c-Src/EGFR transactivation-dependent N-WASP/cdc42/TOCA-1 complex.     

Involvement of sex steroid hormones in the early stages of spermatogenesis in Japanese huchen (Hucho perryi )

In higher vertebrates, considerable progress has been made in understanding the endocrine regulation of puberty; however, in teleosts, the regulatory mechanisms of spermatogenesis during the first annual cycle remain unclear. The present study was conducted to understand the regulatory mechanisms of spermatogenesis throughout the different stages of the first spermatogenic cycle and to check the ability of various steroids and hormones to induce in vitro spermatogonial proliferation in Japanese huchen (Hucho perryi ). The results indicate that the serum level of 11-ketotestosterone (11-KT) was positively associated with germ cell type; the level first began to rise with the appearance of late-type B spermatogonia and continued to increase gradually throughout the active spermatogenic stages and spermiogenesis, reaching a peak value 2 wk before spawning, and then declined. During the spermatogenic stages, the serum concentration of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP) was undetectable. Only a small peak was detected with the appearance of spermatocytes and spermatids, and at the time of spawning, the level increased dramatically, reaching its maximum value with the onset of milt production. Despite the high variation in serum levels of 17beta-estradiol (E2) both between months and among the individuals, E2 was found during the whole reproductive cycle. From these results, we concluded that 1) 11-KT is necessary for the initiation of spermatogenesis and sperm production, and it probably plays a role in spermiation, 2) 17alpha,20beta-DP is essential for the final maturation stage, could play a significant role in the mitosis phase and meiosis process, and probably participates in the regulation of spawning behavior, and 3) estrogen is an indispensable male hormone that plays a physiological role in some aspects of testicular functions, especially during the mitotic phase. The three steroids were also able to induce DNA synthesis, spermatogonial renewal, and/or spermatogonial proliferation in vitro.     

A potential role for caveolin-1 in estradiol-17beta-induced proliferation of mouse embryonic stem cells: involvement of Src, PI3K/Akt, and MAPKs pathways

Although both estrogen and caveolin have been implicated in many physiological functions, their precise relationship is not completely understood in mouse embryonic stem (ES) cells. Thus, this study was designed to examine the relationship between estradiol-17beta (E(2)) and caveolin-1 in mouse ES cell proliferation. E(2) increased the expression of caveolin-1 and caveolin-2 mRNA and proteins, but pre-treatment with ICI 182,780 [an estrogen receptor (ER) antagonist] inhibited E(2)-induced increase in caveolin-1 and caveolin-2 proteins expression. E(2) also increased phosphorylated levels of caveolin-1, Src, and Akt. Phospho-caveolin-1 was significantly blocked by ICI 182,780 or pyrazolopyrimidine 2 (PP2; a Src-kinase inhibitor). LY 294002 (a PI3K inhibitor) or PD 98059 (an ERK1/2 inhibitor) prevented E(2)-induced increase in caveolin-1 expression and the accompanying [(3)H]-thymidine incorporation. Furthermore, inhibition of caveolin-1 expression using a caveolin-1 siRNA significantly attenuated E(2)-induced up-regulation of proto-oncogenes, cell cycle regulatory proteins, [(3)H]-thymidine incorporation, overall cell number, and percent of the cell population in S phase, while mediating a concomitant increase in the G0/G1 population. In conclusion, E(2) stimulates mouse ES cell proliferation partially through up-regulating caveolin-1 via the Src, PI3K/Akt, ERK1/2 signaling pathways.     

Effects of follicle-stimulating hormone and 17β-estradiol on proliferation of chicken embryonic ovarian germ cells in culture

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.     

Effects of follicle-stimulating hormone and 17beta-estradiol on proliferation of chicken embryonic ovarian germ cells in culture

The effects of follicle-stimulating hormone (FSH) and 17beta-estradiol (E2) on chicken ovarian germ cell proliferation were evaluated through a germ-somatic cell coculture model. Ovarian cells from the left ovaries of 18-day-old chicken embryos were cultured in serum-free McCoy's 5A medium at 39 degrees C and challenged with FSH (0.25-1.0 IU/mL) or E2 (10(-8)-10(-5) M) alone and in combination for 48 h. The number of germ cells was counted, and the proliferating cells were immunolocalized by a specific antibody against proliferating cell nuclear antigen (PCNA). The labeling index (LI) was determined for germ cells. Results revealed that germ cells could survive and kept proliferating under support of somatic cells. Germ cells were localized by expression of a specific antibody for stem cell factor receptor c-kit. Both FSH (0.25-1.0 IU/mL) and E2 (10(-7)-10(-5) M) alone induced a marked increase in germ cell number (P<0.05), and PCNA-LI of germ cells was greater in FSH-treated groups (0.25-1.0 IU/mL) and E2-treated groups (10(-8)-10(-5) M), compared with vehicle-treated group (P<0.05). Furthermore, FSH manifested a synergistic effect with E2 (10(-6)-10(-5) M) in stimulating germ cell proliferation. These results indicate that FSH might interact with estrogen to promote ovarian germ cell proliferation in embryonic chickens near hatching.     

Differential alteration of the reproductive axis by testosterone and estrogen in peripubertal and adult male Siberian hamsters (Phodopus sungorus)

In male Siberian hamsters, administration of adult physiological levels of testosterone (T) and estrogen (E2) to juveniles inhibited pubertal onset by distinct pathways. It is presently unclear if T and E2 also exert an inhibitory effect on the reproductive function of sexually mature and sexually maturing hamsters. This study aims to determine if there is an age-dependent decline in the sensitivity of the hypothalamic-pituitary-gonadal (HPG) axis to these inhibitory steroids and if their actions remain distinct. Peripubertal and adult male Siberian hamsters were implanted with a silastic capsule containing T, E2, or cholesterol (Ch, control). Testosterone treatment significantly reduced testes mass and length and impaired spermatogenesis in both ages. In contrast, E2 treatment reduced testes mass only in peripubertal, but not adult, animals. In fact, E2 treatment significantly increased testes mass in adults without altering spermatogenesis. In addition, circulating E2 is very high immediately prior to pubertal onset and declines thereafter. Our results showed the inhibitory effects of T persist into adulthood whereas those of E2 subside as the animals become sexually mature. The decreased sensitivity of the HPG axis to the inhibitory effects of E2 in adult animals and the high level of circulating E2 immediately prior to pubertal onset suggest E2 may play an important role in the regulation of puberty in this species.     

Improved hematopoietic differentiation of human pluripotent stem cells via estrogen receptor signaling pathway

Background

Aside from its importance in reproduction, estrogen (E2) is known to regulate the proliferation and differentiation of hematopoietic stem cells in rodents. However, the regulatory role of E2 in human hematopoietic system has not been investigated. The purpose of this study is to investigate the effect of E2 on hematopoietic differentiation using human pluripotent stem cells (hPSCs).

Results

E2 improved hematopoietic differentiation of hPSCs via estrogen receptor alpha (ER-α)-dependent pathway. During hematopoietic differentiation of hPSCs, ER-α is persistently maintained and hematopoietic phenotypes (CD34 and CD45) were exclusively detected in ER-α positive cells. Interestingly, continuous E2 signaling is required to promote hematopoietic output from hPSCs. Supplementation of E2 or an ER-α selective agonist significantly increased the number of hemangioblasts and hematopoietic progenitors, and subsequent erythropoiesis, whereas ER-β selective agonist did not. Furthermore, ICI 182,780 (ER antagonist) completely abrogated the E2-induced hematopoietic augmentation. Not only from hPSCs but also from human umbilical cord bloods, does E2 signaling potentiate hematopoietic development, suggesting universal function of E2 on hematopoiesis.

Conclusions

Our study identifies E2 as positive regulator of human hematopoiesis and suggests that endocrine factors such as E2 influence the behavior of hematopoietic stem cells in various physiological conditions.

     

Distinct signaling pathways mediate stimulation of cell cycle progression and prevention of apoptotic cell death by estrogen in rat pituitary tumor PR1 cells

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17beta-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones.     

Physiological significance of estrogens in men--breakthrough in endocrinology

Estradiol (E2) is traditionally recognised as the female sex hormone. It has been believed for 40 years, that E2 didn't exert any influence or caused impairment of the gonadal function in men. The main source of E2 in men is adipose tissue and the brain. E2 is also produced in adrenals, liver, mammary glands, hair and in male gonad. Daily production and the level of E2 in the blood in men are higher than those in postmenopausal women. At the end of the 80-ties we were first reporting that during sexual maturation E2 can be the important hormonal signal for the initiation of spermatogenesis. The traditional view about unimportant or inhibitory role of E2 in male physiology was finally refuted thanks to discovering estrogen receptors in males. In the 90-ties, transgenic mice with the lack of estrogen receptor (ER) or gene encoding enzyme aromatase, that enable the conversion of testosterone into E2, were also produced. Observations of men with inherited mutations of these genes, considerably extended our knowledge about E2 in men in stroma bones formation, inhibition of their growing, lipids metabolism and sexual maturation, the effects that were attributed to testosterone action until today. New data also points at important role of estrogens and ER in the function of the cardio-vascular system and in counteracting coronary disease in men.     

Role of HIF-1alpha and VEGF in human mesenchymal stem cell proliferation by 17beta-estradiol: involvement of PKC, PI3K/Akt, and MAPKs

17beta-Estradiol (E(2)) is a steroid hormone well known for its roles in the regulation of various cell functions. However, the precise role that E(2) plays in the proliferation of human mesenchymal stem cells (hMSCs) has not been completely elucidated. In the present study, we examined the effects of E(2) on cell proliferation and the related signaling pathways using hMSCs. We showed that E(2), at > or =10(-9) M, significantly increased [3H]thymidine incorporation after 24 h of incubation, and E(2) also increased [3H]thymidine incorporation at >6 h. Also, E(2) significantly increased the percentage of the cell population in the S phase based on FACS analysis. Moreover, E(2) increased estrogen receptor (ER), PKC, phosphatidylinositol 3-kinase (PI3K)/Akt, and MAPK phosphorylation. Subsequently, these signaling molecules were involved in an E(2)-induced increase of [3H]thymidine incorporation. E(2) also increased hypoxia-inducible factor (HIF)-1alpha and VEGF protein levels. These levels of protein expression were inhibited by ICI-182,780 (10(-6) M, an ER antagonist), staurosporine and bisindolylmaleimide I (10(-6) M, a PKC inhibitor), LY-294002 (10(-6) M, a PI3K inhibitor), Akt inhibitor (10(-5) M), SP-600125 (10(-6) M, a SAPK/JNK inhibitor), and PD-98059 (10(-5) M, a p44/42 MAPKs inhibitor). In addition, HIF-1alpha small interfering (si)RNA and ICI-182,780 inhibited E(2)-induced VEGF expression and cell proliferation. VEGF siRNA also significantly inhibited E(2)-induced cell proliferation. In conclusion, E(2) partially stimulated hMSC proliferation via HIF-1alpha activation and VEGF expression through PKC, PI3K/Akt, and MAPK pathways.     

The importance of hormone receptor analysis in osteosarcoma cells growth submitted to treatment with estrogen in association with thyroid hormone

Bone tumor incidence in women peaks at age 50-60, coinciding with the menopause. That estrogen (E2) and triiodothyronine (T3) interact in bone metabolism has been well established. However, few data on the action of these hormones are available. Our purpose was to determine the role of E2 and T3 in the expression of bone activity markers, namely alkaline phosphatase (AP) and receptor activator of nuclear factor kappaB ligand (RANKL). Two osteosarcoma cell lines: MG-63 (which has both estrogen (ER) and thyroid hormone (TR) receptors) and SaOs-29 (ER receptors only) were treated with infraphysiological E2 associated with T3 at infraphysiological, physiological, and supraphysiological concentrations. Real-time RT-PCR was used for expression analysis. Our results show that, in MG-63 cells, infraphysiological E2 associated with supraphysiological T3 increases AP expression and decreases RANKL expression, while infraphysiological E2 associated with either physiological or supraphysiological T3 decreases both AP and RANKL expression. On the other hand, in SaOs-2 cells, the same hormone combinations had no significant effect on the markers' expression. Thus, the analysis of hormone receptors was shown to be crucial for the assessment of tumor potential growth in the face of hormonal changes. Special care should be provided to patients with T3 and E2 hormone receptors that may increase tumor growth.     

Sertoli-cell prostaglandin synthesis. Effects of (follitropin) differentiation and dietary vitamin E.

The synthesis of prostanoids by the Sertoli cell was assessed as part of a study on the role of vitamin E in maintaining spermatogenesis. Analyses of eicosanoid synthesis from endogenous substrate were carried out using freshly isolated Sertoli-cell-enriched preparations from both pre-pubertal and adult rats fed purified diets with and without vitamin E, as well as cells carried in primary culture. Freshly isolated cells from both the immature and fully differentiated adult testes produced PGI2 (prostaglandin I2) and PGE2, but PGF2 alpha was produced only by cells of the adult vitamin E-deficient rat. Cells from adult controls synthesized PGF2 alpha after primary culture. In contrast with other hormone responses of this cell, which are refractory in the adult, FSH (follitropin) potentiated prostaglandin production by freshly isolated cells of both immature and adult rats. The FSH response of Sertoli cells from immature animals did not change after primary culture. Adult cells were refractory to the hormone after culture, but the total amounts of prostaglandins produced by these cells were 10-fold higher than by either freshly isolated or cells of the immature in culture. Analogues of cyclic AMP did not potentiate prostaglandin synthesis. However, mepacrine, a phospholipase inhibitor, blocked the FSH effect. The finding that Sertoli cells synthesize prostaglandins and FSH enhances prostaglandin production implicates a potential role for eicosanoids in spermatogenesis and suggests that vitamin E may affect intratesticular regulators.     

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells In Vitro and In Vivo

Use of estrogen or estrogen / progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD in vitro. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment in vitro and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth in vivo. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.     

Review of <Emphasis Type="Italic">Molecular Biology of Human Cancers</Emphasis> (<Emphasis Type="Italic">An Advanced Student’s Text</Emphasis>, by Wolfgang Schulz (Department of Urology and Center for Biological and Medical Research,Heinrich Heine University,Düsseldorf) ISBN 1-4020-3185-8

Postmenopausal women with estrogen receptor positive (ER⁺) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17 -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER⁺ postmenopausal breast cancer cells.     

Down-regulation of Bcl-2 enhances estrogen apoptotic action in long-term estradiol-depleted ER<Superscript>+</Superscript> breast cancer cells

Postmenopausal women with estrogen receptor positive (ER⁺) breast cancer frequently respond paradoxically to estrogen administration with tumor regression. Using both LTED and E8CASS cells derived from MCF-7 breast cancer cells by long-term estrogen-deprivation, we previously reported that 17 -estradiol (estradiol) is a powerful, pro-apoptotic hormone which kills the cancer cells through activation of the Fas/FasL death receptor pathway. We postulated that the mitochondrial interactive protein Bcl-2 might play a role in the regulation of estradiol-induced apoptosis in both LTED and E8CASS cells. In this study, we assessed estradiol effects on cell growth, proliferation and apoptosis. Additionally we investigated the effect of estradiol on caspase activation, NF-KB and Bcl-2 expression. The functional role of Bcl-2 in estradiol-induced apoptosis was further studied by knockdown or decrease of Bcl-2 with siRNA. Our results show that estradiol significantly inhibited cell growth primarily through a pro-apoptotic action involving caspase-7 and 9 activations (p < 0.01). Basal Bcl-2 and NF-KB levels were greatly elevated and estradiol decreased NF-KB, but not Bcl-2 expression. Knockdown of Bcl-2 expression with siRNA decreased the levels of this protein by 9 fold (p < 0.01). This reduction markedly sensitized both LTED and E8CASS cells to the pro-apoptotic action of estradiol, leading to a synergistic induction of apoptosis and a concomitant reduction in cell number (p < 0.01). Therefore, down-regulation of Bcl-2 synergistically enhanced estradiol-induced apoptosis in ER⁺ postmenopausal breast cancer cells.