Estrogen-induced Apoptosis Research Articles

Abstract 3924: Alteration of the shape of the ligand estrogen receptor complex controls estrogen induced apoptosis in breast cancer

Abstract Aromatase inhibitors (AIs) are the standard of care for long term adjuvant treatment of estrogen receptor (ER) positive breast cancer in postmenopausal patients. However, acquired drug resistance eventually develops and evolves to expose a vulnerability in breast cancer: estrogen induced apoptosis. The discovery of the new biology of estrogen induced apoptosis has translated to treatment of patients in clinical trials [Jordan V, Ford L. Cancer Prev Res 2011;4:633-7]. We recently reported the sequence of events involved in triggering of estrogen induced apoptosis [Ariazi et al. Proc Natl Acad Sci USA. 2011]. Our goal here is to define the mechanics of ligand ER action to trigger apoptosis. 4-Hydroxytamoxifen (4OHT) reverses estradiol (E2) induced apoptosis in long term estrogen deprived MCF-7 cells. As an investigative tool, we synthesized a range of estrogenic triphenylethylenes (TPEs), which are structurally similar to 4OHT. We discovered that although the TPEs are all classified as estrogens for breast cancer growth they, like 4OHT, inhibit E2 induced apoptosis. We previously identified that the surface amino acid D351 within the ligand binding domain is critical for the estrogenic actions of 4OHT. Unlike raloxifene, whose antiestrogen side chain shields and neutralizes D351, the side chain in 4OHT is too short. In order to elucidate the biological activity of the TPE:ER, we devised an assay using induction of the mRNA for the transforming growth factor (TGF-β) gene in situ[Jordan et al. Cancer Res. 2001;61:6619-6623]. In MDA-MB231 cells stably transfected with wild type ER (MC2 cells), TPEs and tamoxifen metabolites, 4OHT and endoxifen induce TGF- ≤ mRNA expression in a similar fashion to 17α estradiol (E2). Cells stably expressing D351G ER (JM6 cells) i.e. without the charged D351, ERα retain responsiveness to planar estrogens; however the nonplanar estrogens and 4OHT, lose their ability to induce TGF-β mRNA and become completely antiestrogenic. This response therefore is characteristic of an antiestrogenic conformation of the ER complex where charged D351 would interact with activating function 1(AF-1) to enhance estrogen action for growth. The TPEs prefer to dock with the ER in a conformation related to 4OHT: the antiestrogenic conformation. The fact that the TPEs also stimulate growth of breast cancer demonstrates the extreme sensitivity of this estrogenic endpoint. Thus the observation that planar and nonplanar compounds have different abilities to change the shape of the ER complex may have important implication for the modulation of the E2 induced apoptosis. We conclude that AF-1 is required for E2 induced growth but AF1 and AF2 together are required to deliver coactivators and cause E2 induced apoptosis. Acknowledgements This work (VCJ) was supported by the Susan G Komen for the Cure Foundation under Award number SAC100009. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3924. doi:1538-7445.AM2012-3924

Abstract 786: In-vivo and in-vitro breast cancer profiling to delineate adaptive endocrine resistance: A pilot study

Abstract A substantial fraction of estrogen receptor (ER) positive breast cancers fail to respond to endocrine treatment, i.e. tamoxifen and aromatase inhibitors. While tamoxifen failure has been partially attributed to primary resistance, for example CYP2D6 deficiency (Schroth et al JAMA 2009), adaptive resistance during ER blockade/estrogen deprivation plays a prominent role. Little is known about shared molecular patterns driving both resistance types. Long-term estrogen deprived (LTED) breast cancer can be induced to undergo apoptosis by pulse estrogen (Song et al JNCI 2001; Jordan JCO 2008). We hypothesize that it should be possible to identify predictive markers for adaptive endocrine resistance and sensitivity to apoptosis to recognize endocrine refractory tumors. To study a putative interface of primary and acquired resistance we performed gene expression profiling with low-density arrays (Fluidigm BioMark system) in tumors of 12 breast cancer patients treated with tamoxifen. They were selected to model CYP2D6 independent primary resistance: half of the patients had functional CYP2D6 but were tamoxifen resistant due to relapse (median time-to-event 3.8 years). The other half of patients were CYP2D6 deficient but tamoxifen sensitive due to the absence of relapse (median follow-up 13.5 years). Expression profiles have been compared to LTED MCF-7 cells (3 and 6 months estrogen deprived) and MCF-7 cells grown in the presence of estrogen thereby modeling adaptive resistance. Gene targets were selected based on literature i) involved in endocrine resistance (N=62), ii) playing a role in transport and estrogen supply (N=30), and iii) being p53 targets for apoptosis and cell-cycle check points (N=46). We observed both shared and specific expression phenotypes for adaptive and primary resistance. The proliferation genes Cyclin B1 and STK15 were downregulated in LTED MCF-7 cells in line with lowered proliferation rates and, unexpectedly, Cyclin B1 expression was also decreased in tamoxifen insensitive tumors suggesting an involvement of G2/M cell cycle transition. The chemokine receptor CXCR4 was upregulated in both LTED cells and resistant tumors, confirming its prominent role in breast cancer progression. The 25-hydroxyvitamin D3 hydroxylase CYP27B1 was upregulated in LTED, but not in resistant tumors suggesting a different role between the two models. Other potential markers involve differentiation, inflammatory response, cell cycle/apoptosis, and estrogen disposition pathways. Our preliminary data demonstrate the feasibility to discern between short-term adaptive versus primary resistance patterns. Expression profiling of LTED cells following estrogen-induced apoptosis is currently underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 786. doi:1538-7445.AM2012-786

Paradoxical Clinical Effect of Estrogen on Breast Cancer Risk: A “New” Biology of Estrogen-induced Apoptosis

Administration of estrogen replacement therapy (ERT) decreases the incidence of breast cancer, as shown in a double-blind, placebo-controlled randomized trial of the Women's Health Initiative (WHI) in 10,739 postmenopausal women with a prior hysterectomy. Although paradoxical because estrogen is recognized to stimulate breast cancer growth, laboratory data support a mechanism of estrogen-induced apoptosis under the correct environmental circumstances. Long-term antiestrogen treatment or estrogen deprivation causes the eventual development and evolution of antihormone resistance. Cell populations emerge with a vulnerability, as estrogen is no longer a survival signal but is an apoptotic trigger. The antitumor effect of ERT in estrogen-deprived postmenopausal women is consistent with laboratory models.

The conformation of the estrogen receptor directs estrogen-induced apoptosis in breast cancer: a hypothesis

BACKGROUND: Estrogens are classified as type I (planar) and type II (angular) based on their structures. In this study we have used triphenylethylenes (TPEs) compounds related to 4OHT to address the hypothesis that the conformation of the liganded estrogen receptor (ERα) may dictate the E2-induced apoptosis of the ER+ breast cancer cells. MATERIALS AND METHODS: ERα positive MCF7:5C cells were used to study the apoptosis induced by E2, 4OHT and TPEs. Growth and apoptosis assay were used to evaluate apoptosis and the ability to reverse the E2-induced apoptosis. ERα protein were measured by western blotting to investigate the destruction of ERα by TPEs in MCF7 cells. ChIP assay were performed to study the in-vivo recruitment of ERα and SRC3 at classical E2-responsive promoter TFF1 (PS2) by TPEs. Molecular modeling was used to predict the binding mode of the TPE to the ERα. RESULTS: TPEs were not only unable to induce efficient apoptosis in MCF7:5C cells but also reversed the E2-induced apoptosis similar to 4OHT. Furthermore, the TPEs and 4OHT did not reduce the ERα protein levels unlike E2. ChIP assay confirmed very weak recruitment of SRC3 despite modest recruitment of ERα in the presence of TPEs. Molecular modeling suggested the TPE would bind in antagonistic mode with the ERα. CONCLUSION: Our results advances the hypothesis that the TPE liganded ERα complex structurally resembles the 4OHT bound ERα and cannot efficiently recruit co-activator SRC3. As a result, the TPE complex cannot induce apoptosis of ER+ breast cancer cells although it may cause growth of the breast cancer cells. The conformation of the estrogen-ER complex differentially controls growth and apoptosis.

Estrogen-Induced Apoptosis of Breast Epithelial Cells Is Blocked by NO/cGMP and Mediated by Extranuclear Estrogen Receptors

Estrogen action, via both nuclear and extranuclear estrogen receptors (ERs), induces a variety of cellular signals that are prosurvival or proliferative, whereas nitric oxide (NO) can inhibit apoptosis via caspase S-nitrosylation and via activation of soluble guanylyl cyclase to produce cGMP. The action of 17β-estradiol (E(2)) at ER is known to elicit NO signaling via activation of NO synthase (NOS) in many tissues. The MCF-10A nontumorigenic, mammary epithelial cell line is genetically stable and insensitive to estrogenic proliferation. In this cell line, estrogens or NOS inhibitors alone had no significant effect, whereas in combination, apoptosis was induced rapidly in the absence of serum; the presence of inducible NOS was confirmed by proteomic analysis. The application of pharmacological agents determined that apoptosis was dependent upon NO/cGMP signaling via cyclic GMP (cGMP)-dependent protein kinase and could be replicated by inhibition of the phosphatidylinositol 3 kinase/serine-threonine kinase pathway prior to addition of E(2). Apoptosis was confirmed by nuclear staining and increased caspase-3 activity in E(2) + NOS inhibitor-treated cells. Apoptosis was partially inhibited by a pure ER antagonist and replicated by agonists selective for extranuclear ER. Cells were rescued from E(2)-induced apoptosis after NOS blockade, by NO-donors and cGMP pathway agonists; preincubation with NO donors was required. The NOS and ER status of breast cancer tissues is significant in etiology, prognosis, and therapy. In this study, apoptosis of preneoplastic mammary epithelial cells was triggered by estrogens via a rapid, extranuclear ER-mediated response, after removal of an antiapoptotic NO/cGMP/cGMP-dependent protein kinase signal.

Abstract B61: Phospholipid scramblase is a critical mediator of estrogen-induced apoptosis in antihormone resistant breast cancer cells

Abstract Phospholipid scramblase 1 (PLSCR1) is a mutiply palmitoylated protein which is localized in either the cell membrane or nucleus depending on its palmitoylated state and is responsible for the translocation of phospholipids between the two monolayers of a lipid bilayer of a cell membrane in a calcium (Ca2+)-dependent manner. There is evidence that PLSCR1 plays a role in cell signaling and apoptosis. In the present study, we investigated the functions of PLSCR1 in hormone-sensitive MCF-7 and T47D breast cancer cells versus antihormone-resistant MCF-7:5C and MCF-7:2A cells. We found that PLSCR1 protein and mRNA levels were dramatically increased in MCF-7:5C and MCF-7:2A cells compared to parental MCF-7 and T47D cells and suppression of PLSCR1 in these cells using small interfering RNA (siRNA) reduced their growth rate and altered their morphology to become more differentiated and spindle-shaped. Suppression of PLSCR1 also completely blocked estrogen-induced apoptosis in MCF-7:5C cells. Furthermore, we found that stable expression of PLSCR1 in MCF-7 cells enhanced their sensitivity to etoposide-induced apoptosis and that treatment of these cells with interferon-alpha 2 (IFN- 2) resulted in cell cycle arrest. Overall, these data show that PLSCR1 is overexpressed in antihormone-resistant breast cancer cells and they suggest that PLSCR1 plays an important role in sensitizing these cells to estrogen-induced apoptosis. These data also shed new insights into the biological and biochemical functions of PLSCR1 in breast cancer. Citation Information: Cancer Epidemiol Biomarkers Prev 2010;19(10 Suppl):B61.

Direct effects of estrogen on differentiation and apoptosis of osteoclasts

Osteoclasts play a crucial role in bone resorption. Since osteoclast differentiation/activation is involved in orthodontic tooth movement at the compression sites, the investigation on osteoclasts is very important to the field of orthodontics. It is well known that estrogen has the protective effect on bone. However, the mechanisms by which estrogen prevents bone loss remain to be elucidated. Although estrogen was recently reported to induce apoptosis of osteoclasts, the precise mechanisms of estrogen-induced osteoclast apoptosis remained controversial with regard to whether estrogen affects osteoclasts directly or not. Here we investigated whether estrogen directly induces differentiation and apoptosis of osteoclasts in vitro using mouse monocytic RAW264 cells differentiated into osteoclasts by RANKL. It was observed that estrogen inhibited RANKL-induced osteoclast differentiation of RAW264 cells in a dose-dependent manner. Estrogen suppressed p38 phosphorylation while it enhanced ERK phosphorylation induced by RANKL, suggesting that modulation of MAPK signaling may be involved in inhibition of osteoclast differentiation by estrogen. Next, it was shown that estrogen dose-dependently augmented caspase-3 activation in osteoclasts differentiated from RAW264 cells by RANKL, demonstrating that estrogen directly enhanced apoptosis of osteoclasts. Estrogen-induced caspase-3 activation was attenuated by ICI 182,780, suggesting that the effects of estrogen on osteoclast apoptosis is mediated through estrogen receptors. Thus, these results suggest that estrogen may directly inhibit differentiation and induce apoptosis of osteoclasts.

The synergistic effect of Mig-6 and Pten ablation on endometrial cancer development and progression.

Ablation of Mig-6 in the murine uterus leads to the development of endometrial hyperplasia and estrogen-induced endometrial cancer. An additional endometrial cancer mouse model is generated by ablation of Pten (either as heterozygotes or by conditional uterine ablation). To determine the interplay between Mig-6 and the PTEN/PI3K signaling pathway during endometrial tumorigenesis, we have generated mice with Mig-6 and Pten conditionally ablated in progesterone receptor positive cells (PRcre/+Mig-6f/fPtenf/f ; Mig-6d/dPtend/d). The ablation of both Mig-6 and Pten dramatically accelerated the development of endometrial cancer compared to single ablation of either gene. The epithelium of Mig-6d/dPtend/d mice showed a significant decrease in the number of apoptotic cells compared to Ptend/d mice. The expression of the estrogen-induced apoptotic inhibitors Birc1 was significantly increased in the Mig-6d/dPtend/d mice. We identified ERK2 as a MIG-6 interacting protein by co-immunoprecipitation and demonstrated that the level of ERK2 phosphorylation was increased upon Mig-6 ablation either singly or in combination with Pten ablation. These results suggest that Mig-6 exerts a tumor suppressor function in endometrial cancer by promoting epithelial cell apoptosis through the down-regulation of the estrogen-induced apoptosis inhibitors Birc1 and the inhibition of ERK2 phosphorylation.

Abstract 1279: Caspase-4 is critical in estrogen-induced apoptosis in antihormone-resistant breast cancer

Abstract Acquired resistance to long-term antihormonal therapy in breast cancer evolves through two Phases over 5 years. Phase I develops within a year, and tumor growth occurs with either estradiol or tamoxifen. However, Phase II resistance develops after 5 years of therapy. Tamoxifen continues to stimulate tumor growth, however the resistant tumor cell evolves so that estradiol is no longer a survival signal, but instead induces apoptosis. This is the basis for the therapeutic use of estrogen to treat breast cancer. Unfortunately only 30% of tumors respond, so laboratory studies were designed to seek mechanisms to enhance apoptotic responses. We have compared the actions of estradiol in MCF-7 variants that are invulnerable to estradiol-induced apoptosis (wild-type WS8), rapidly induce apoptosis (5C) or exhibit delayed apoptosis (2A). Using Agilent 4×44k human oligonucleotide microarrays, gene expression profiling of these cell lines treated with and without E2 was conducted over a 96 hour time course. Additionally, a long-term 9 day time course of 2A cells was conducted to capture its delayed apoptotic response. A statistically rigorous method was developed to analyze the time course expression profiles in which the area under the curve (AUC) was computed for all genes and compared between cell lines. Focusing on apoptotic genes, caspase-4 was the earliest caspase significantly induced in the 5C cells from 24-96 h, and in the 2A cells from 6-9 days. Caspase-4 is specifically associated with the accumulation of unfolded proteins and protein aggregation in endoplasmic reticulum stress-induced apoptosis. Consistent with this mechanism of apoptosis, the observed gene expression profiles indicated a lack of induction in both resistant cell lines compared to wild-type cells of genes involved in protein folding such as chaperones (e.g. HSPA13, HSP70 member 13), co-chaperones (e.g. AHSA1, activator of HSP90 protein ATPase homolog 1) and chaperonins (HSP60/HPSD1; TCP1, T-complex 1). Additionally, the pro-apoptotic factor Bim, which is also associated with endoplasmic reticulum stress, was induced in both resistant cell lines compared to wild-type cells. Caspase-4 induction was confirmed by quantitative PCR and immunoblotting. The specific caspase-4 inhibitor Z-LEVD-FMK not only reduced cleaved caspase 4, it also completely blocked PARP cleavage, and estradiol-induced apoptosis changes in morphology and growth. Based on these data, a strategy to sensitize cells to estradiol-induced apoptosis could be applied to improve clinical response rates in Phase II acquired resistance to antihormone therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1279.

Estrogen regulation of apoptosis: how can one hormone stimulate and inhibit?

The link between estrogen and the development and proliferation of breast cancer is well documented. Estrogen stimulates growth and inhibits apoptosis through estrogen receptor-mediated mechanisms in many cell types. Interestingly, there is strong evidence that estrogen induces apoptosis in breast cancer and other cell types. Forty years ago, before the development of tamoxifen, high-dose estrogen was used to induce tumor regression of hormone-dependent breast cancer in post-menopausal women. While the mechanisms by which estrogen induces apoptosis were not completely known, recent evidence from our laboratory and others demonstrates the involvement of the extrinsic (Fas/FasL) and the intrinsic (mitochondria) pathways in this process. We discuss the different apoptotic signaling pathways involved in E2 (17β-estradiol)-induced apoptosis, including the intrinsic and extrinsic apoptosis pathways, the NF-κB (nuclear factor-kappa-B)-mediated survival pathway as well as the PI3K (phosphoinositide 3-kinase)/Akt signaling pathway. Breast cancer cells can also be sensitized to estrogen-induced apoptosis through suppression of glutathione by BSO (L-buthionine sulfoximine). This finding has implications for the control of breast cancer with low-dose estrogen and other targeted therapeutic drugs.

Potential of l-buthionine sulfoximine to enhance the apoptotic action of estradiol to reverse acquired antihormonal resistance in metastatic breast cancer

l-Buthionine sulfoximine (BSO) is a potent inhibitor of glutathione biosynthesis and studies have shown that it is capable of enhancing the apoptotic effects of several chemotherapeutic agents. Previous studies have shown that long-term antihormonal therapy leads to acquired drug resistance and that estrogen, which is normally a survival signal, is a potent apoptotic agent in these resistant cells. Interestingly, we have developed an antihormone-resistant breast cancer cell line, MCF-7:2A, which is resistant to estrogen-induced apoptosis but has elevated levels of glutathione. In the present study, we examined whether BSO is capable of sensitizing antihormone-resistant MCF-7:2A cells to estrogen-induced apoptosis. Our results showed that treatment of MCF-7:2A cells with 1 nM E2 plus 100 μM BSO combination for 1 week reduced the growth of these cells by almost 80–90% whereas the individual treatments had no significant effect on growth. TUNEL and 4′,6-diamidino-2-phenylindole (DAPI) staining showed that the inhibitory effect of the combination treatment was due to apoptosis. Our data indicates that glutathione participates in retarding apoptosis in antihormone-resistant human breast cancer cells and that depletion of this molecule by BSO may be critical in predisposing resistant cells to estrogen-induced apoptosis.

Buthionine sulfoximine sensitizes antihormone-resistant human breast cancer cells to estrogen-induced apoptosis

IntroductionEstrogen deprivation using aromatase inhibitors is one of the standard treatments for postmenopausal women with estrogen receptor (ER)-positive breast cancer. However, one of the consequences of prolonged estrogen suppression is acquired drug resistance. Our group is interested in studying antihormone resistance and has previously reported the development of an estrogen deprived human breast cancer cell line, MCF-7:5C, which undergoes apoptosis in the presence of estradiol. In contrast, another estrogen deprived cell line, MCF-7:2A, appears to have elevated levels of glutathione (GSH) and is resistant to estradiol-induced apoptosis. In the present study, we evaluated whether buthionine sulfoximine (BSO), a potent inhibitor of glutathione (GSH) synthesis, is capable of sensitizing antihormone resistant MCF-7:2A cells to estradiol-induced apoptosis.MethodsEstrogen deprived MCF-7:2A cells were treated with 1 nM 17β-estradiol (E2), 100 μM BSO, or 1 nM E2 + 100 μM BSO combination in vitro, and the effects of these agents on cell growth and apoptosis were evaluated by DNA quantitation assay and annexin V and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) staining. The in vitro results of the MCF-7:2A cell line were further confirmed in vivo in a mouse xenograft model.ResultsExposure of MCF-7:2A cells to 1 nM E2 plus 100 μM BSO combination for 48 to 96 h produced a sevenfold increase in apoptosis whereas the individual treatments had no significant effect on growth. Induction of apoptosis by the combination treatment of E2 plus BSO was evidenced by changes in Bcl-2 and Bax expression. The combination treatment also markedly increased phosphorylated c-Jun N-terminal kinase (JNK) levels in MCF-7:2A cells and blockade of the JNK pathway attenuated the apoptotic effect of E2 plus BSO. Our in vitro findings corroborated in vivo data from a mouse xenograft model in which daily administration of BSO either as a single agent or in combination with E2 significantly reduced tumor growth of MCF-7:2A cells.ConclusionsOur data indicates that GSH participates in retarding apoptosis in antihormone-resistant human breast cancer cells and that depletion of this molecule by BSO may be critical in predisposing resistant cells to E2-induced apoptotic cell death. We suggest that these data may form the basis of improving therapeutic strategies for the treatment of antihormone resistant ER-positive breast cancer.

Estrogen protects bone by inducing Fas ligand in osteoblasts to regulate osteoclast survival

Estrogen deficiency in menopause is a major cause of osteoporosis in women. Estrogen acts to maintain the appropriate ratio between bone-forming osteoblasts and bone-resorbing osteoclasts in part through the induction of osteoclast apoptosis. Recent studies have suggested a role for Fas ligand (FasL) in estrogen-induced osteoclast apoptosis by an autocrine mechanism involving osteoclasts alone. In contrast, we describe a paracrine mechanism in which estrogen affects osteoclast survival through the upregulation of FasL in osteoblasts (and not osteoclasts) leading to the apoptosis of pre-osteoclasts. We have characterized a cell-type-specific hormone-inducible enhancer located 86 kb downstream of the FasL gene as the target of estrogen receptor-alpha induction of FasL expression in osteoblasts. In addition, tamoxifen and raloxifene, two selective estrogen receptor modulators that have protective effects in bone, induce apoptosis in pre-osteoclasts by the same osteoblast-dependent mechanism. These results demonstrate that estrogen protects bone by inducing a paracrine signal originating in osteoblasts leading to the death of pre-osteoclasts and offer an important new target for the prevention and treatment of osteoporosis.

Mechanism of estrogen-induced apoptosis in breast cancer cells: Role of the NF-κB signaling pathway

The ability of sex steroid hormones to up-regulate the apoptotic signaling proteins is well documented; however, the apoptotic potential of sex hormones is not remarkable and fully compensated by their growth stimulatory action to target cells. In the present study using the long-term cultivation of estrogen-dependent MCF-7 breast cancer cells in steroid-free medium, we have established a cell subline, designed as MCF-7/LS, which was characterized by the resistance to growth stimulatory estradiol action and hypersensitivity to estrogen-induced apoptosis. We have demonstrated that estrogen treatment of the cells does not influence on the level of TNF-R1 or Fas, but dramatically decreases the transcriptional activity of NF-kappaB. Importantly, the MCF-7/LS cells, which are insensitive to growth stimulatory estrogen action, retain the ability to decrease in the NF-kappaB activity in response to estrogen stimulus. Furthermore, the significant increase in the basal (in the absence of ligand) estrogen receptor (ER)-dependent transcriptional activity in the MCF-7/LS cells was revealed and reciprocal transcriptional antagonism between ER and NF-kappaB was demonstrated. Finally, we proved the possible involvement of phosphatidylinositol-3 kinase (PI3K) in the ligand-independent ER activation. In general, the results presented suggest that long-term growth of MCF-7 breast cancer cells in steroid-free medium is accompanied with the increase in the basal ER-dependent transcriptional activity as well as the maintenance of the negative regulatory loop ER-NF-kappaB. The latter may be considered as one of the factors resulting in a disbalance between pro- and anti-apoptotic pathways and enhancement in estrogen apoptotic action in the cells.

Activation of mitogenic pathways and sensitization to estrogen-induced apoptosis: two independent characteristics of tamoxifen-resistant breast cancer cells?

Paradoxical induction of apoptosis by estrogen has been described previously for estrogen-deprived and antiestrogen-resistant breast cancer cells. In this study we analyzed the possible interrelations between cell sensitization to estrogen apoptotic action and cell ability to (anti)estrogen-independent growth. Using tamoxifen-resistant sublines derived from the parent MCF-7 breast cancer cells by long-term tamoxifen treatment we demonstrated that resistant cells are characterized by increased level of EGF receptor and unexpected increase of VEGF receptor 2 (Flk-1/KDR) and its specific ligand, VEGF-A. The importance of the VEGF signaling in the autocrine regulation of cell growth was indicated by the ability of VEGF inhibitor, soluble fragment of Flt-1/Fc chimera, to suppress the phosphorylation of MAP kinases as well as to inhibit the estrogen-independent growth of MCF-7 cells. Sensitization of tamoxifen-resistant cells to estrogen-induced apoptosis required the additional continuous cultivation in steroid-depleted medium and did not depend on the activity of both EGF and VEGF pathways. Finally, we showed that treatment of the cells with 17beta-estradiol (10(-9) M) resulted in a marked increase in p53 level both in the resistant cells undergoing apoptosis and in the parent MCF-7 cells insensitive to apoptotic estrogen action. These data provide an important support for the existence of a disbalance between pro- and anti-apoptotic machinery in the resistant breast cancer cells that forms independently of the acquired ability to estrogen-independent growth.

Sensitization of MCF-7 breast cancer cells to the apoptotic effect of estradiol

A new substrain of hormone-resistant MCF-7/T breast cancer cells was selected after long-term culturing of estrogen-dependent MCF-7 cells in the presence of tamoxifen. These cells were resistant to the growth-stimulating and cytostatic effects of estradiol and tamoxifen, respectively. MCF-7/T cells gained paradoxical sensitivity to the apoptotic effect of estradiol. Estradiol stimulated p53 expression and decreased DNA-binding activity of NF-kappaB. Our findings provide indirect evidence that these proteins are involved in the regulation of estrogen-induced apoptosis. These results indicate that tamoxifen-resistant breast cancer cells can be sensitized to the apoptotic effect of estradiol. The data form a basis for the development of new methods of endocrine therapy for breast cancer patients.

17β-Estradiol Induces Apoptosis in the Developing Rodent Prostate Independently of ERα or ERβ

Estrogens induce both proliferative and antiproliferative responses in the prostate gland. To date, antiproliferative effects of estrogens are generally considered to be due to systemic antiandrogenic actions. However, estrogen action mediated through estrogen receptor (ER) beta was recently suggested as another mechanism of induction of apoptosis in the prostate. This study aimed to explore the hypothesis that the antiproliferative effects of estrogen are directly mediated through ERbeta using a prostate organ culture system. We previously reported effects of 17beta-estradiol (E2) using rat ventral prostate (VP) tissues, and adapted the system for culturing mouse tissues. In both rat and mouse models, estrogen-induced apoptosis was detected that was spatially and regionally localized to the epithelium of the distal tips. Using organ cultures of alphaER knockout (alphaERKO) and betaERKO prostates, we failed to demonstrate that apoptosis induced by E2 was mediated through either receptor subtype. Activation of ER-selective ligands (ERalpha, propyl pyrazole triol, ERbeta, diaryl-proprionitrile, and 5alpha-androstane-3beta,17beta-diol) in organ culture experiments failed to induce apoptosis, as did the membrane impermeable conjugate E2:BSA, discounting the possibility of nongenomic effects. Consequently, E2 regulation of androgen receptor (AR) expression was examined and, in the presence of nanomolar testosterone levels, E2 caused a specific reduction in AR protein expression in wild-type, alphaERKO, and betaERKO mice, particularly in the distal region where apoptosis was detected. This down-regulation of AR protein provides a possible mechanism for the proapoptotic action of E2 that is independent of ERs or nongenomic effects.

Targeting estrogen to kill ER-positive and ER-negative breast cancer

The current fashion of using long-term antihormonal therapies for the treatment and prevention of breast cancer has been remarkably successful over the past 20 years but this strategy has consequences for the development of drug resistance in remaining tumor tissue. Although estrogen is considered to be a survival signal that causes increased breast cancer cell replication, the study of drug resistance to antihormonal therapies has revealed an unanticipated new biology of estrogen action. Long-term antihormonal therapy eventually results in either tamoxifen or raloxifene (selective estrogen receptor modulators [SERMs]) stimulated growth and tumors are also stimulated to grow with estrogen. This is why aromatase inhibitors are effective treatments after the development of SERM resistance once the SERM is stopped. Long-term estrogen deprivation initially causes a cessation of breast tumor cell growth but eventually cells grow out that remain ER-positive but grow spontaneously. Estrogen deprivation with SERMs or aromatase inhibitors for more than 5 years causes a remarkable switching of the estrogen signaling pathway [1]. Instead of being a survival signal, physiologic concentrations of estrogen now cause apoptosis and tumor cell death. This knowledge provides an opportunity to test the hypothesis that low-dose estrogen therapy following exhaustive antihormonal therapy could be used as a successful treatment for patients. Studies are in place to evaluate the mechanism of action of estrogen-induced apoptosis so that a new target can be discovered to develop a novel apoptotic drug group. The ER-negative breast cancer cell is the ultimate hormone-resistant cell. Reintroduction of an active ER gene re-sensitizes the cells to estrogen that now causes blockade of the cell cycle [2] and apoptosis if cell survival signaling is also blocked. These data suggest that a universal target could be identified using the estrogen receptor mediated mechanism that will permit the broad application of new anti-apoptotic medicines.

Estrogen-induced apoptosis in a breast cancer model resistant to long-term estrogen withdrawal

Estrogen suppression through the use of an aromatase inhibitor is an effective endocrine treatment option for postmenopausal breast cancer patients with estrogen receptor (ER)-positive disease, however, there are concerns that long-term estrogen deprivation will inevitably lead to resistance. To address the issue of acquired resistance to long-term estrogen deprivation our laboratory has developed an ER+/PR− hormone-independent breast cancer cell line, MCF-7:5C which is a variant clone of wild-type MCF-7 cells. Originally, these cells were cultured in estrogen-free MEM containing 5% charcoal-stripped calf serum and were found to be resistant to both estradiol (E 2) and antiestrogens. Interestingly, a completely different phenomenon was observed when MCF-7:5C cells were cultured in phenol red-free RPMI 1640 medium containing 10% charcoal-stripped fetal bovine serum (SFS). Using DNA quantitation assays, we examined the effect of E 2 on the growth of MCF-7:5C cells under different media conditions. Our results showed that 10 −9 M E 2 caused a dramatic 90% reduction in the growth of MCF-7:5C cells cultured in RPMI medium containing 10% SFS but did not have any significant inhibitory effects on cells cultured in MEM media. Additional experiments were performed to determine whether the medium or the serum facilitated the inhibitory effects of E 2 and the results indicated that it was the serum. Annexin V and DAPI staining confirmed that the E 2-induced growth inhibition of MCF-7:5C cells was due to apoptosis. We also examined the tumorigenic potential of MCF-7:5C cells by injecting 1 × 10 7 cells/site into ovariectomized athymic mice and found that these cells, previously cultured in RPMI media, spontaneously grew into tumors in the absence of E 2. Overall, these results show that low concentrations (>10 −11 M) of E 2 are capable of inducing apoptosis in an aromatase resistant breast cancer cell model and that this effect is highly influenced by the medium in which the cells are grown.

Estrogen-induced Spermatogenic Cell Apoptosis Occurs via the Mitochondrial Pathway: ROLE OF SUPEROXIDE AND NITRIC OXIDE

The detrimental effects of estrogen on testicular function provide a conceptual basis to examine the speculative link between increased exposure to estrogens and spermatogenic cell death. Using an in vitro model, we provide an understanding of the events leading to estrogen-induced apoptosis in cells of spermatogenic lineage. Early events associated with estrogen exposure were up-regulation of FasL and increased generation of H(2)O(2), superoxide, and nitric oxide. The ability of anti-FasL antibodies to prevent several downstream biochemical changes and cell death induced by 17beta-estradiol substantiates the involvement of the cell death receptor pathway. Evidence for the amplification of the death-inducing signals through mitochondria was obtained from the transient mitochondrial hyperpolarization observed after estradiol exposure resulting in cytochrome c release. A combination of nitric oxide and superoxide but not H(2)O(2) was responsible for the mitochondrial hyperpolarization. Mn(III) tetrakis(4-benzoic acid)porphyrin chloride, an intracellular peroxynitrite scavenger, was able to reduce mitochondrial hyperpolarization and cell death. Although nitric oxide augmentation occurred through an increase in the expression of inducible nitric-oxide synthase, superoxide up-regulation was a product of estradiol metabolism. All of the above changes were mediated through an estrogen receptor-based mechanism because tamoxifen, the estrogen receptor modulator, was able to rescue the cells from estrogen-induced alterations. This study establishes the importance of the independent capability of cells of the spermatogenic lineage to respond to estrogens and most importantly suggests that low dose estrogens can potentially cause severe spermatogenic cellular dysfunction leading to impaired fertility even without interference of the hypothalamo-hypophyseal axis.