ERE-luciferase Reporter Assay Research Articles

FOXC1 is associated with estrogen receptor alpha and affects sensitivity of tamoxifen treatment in breast cancer.

FOXC1 is a member of Forkhead box transcription factors that participates in embryonic development and tumorigenesis. Our previous study demonstrated that FOXC1 was highly expressed in triple‐negative breast cancer. However, it remains unclear what is the relation between FOXC1 and ERα and if FOXC1 regulates expression of ERα. To explore relation between FOXC1 and ERα and discover regulation of ERα expression by FOXC1 in breast cancer, we analyzed data assembled in the Oncomine and TCGA, and found that there was significantly higher FOXC1 expression in estrogen receptor‐negative breast cancer than that in estrogen receptor‐positive breast cancer. Overexpression of FOXC1 reduced expression of ERα and cellular responses to estradiol (E2) and tamoxifen in the MCF‐7 FOXC1 and T47D FOXC1 cells, while knockdown of FOXC1 induced expression of ERα and improved responses to estradiol (E2) and tamoxifen in BT549 FOXC1 shRNA and HCC1806 FOXC1 shRNA cells. In addition, overexpression of FOXC1 reduced expression of progesterone receptor (PR), Insulin receptor substrate 1 (IRS1), and XBP1 (X‐Box Binding Protein 1) and significantly reduced luciferase activity caused by E2 using ERE luciferase reporter assay. These results suggested that FOXC1 regulated expression of ERα and affected sensitivity of tamoxifen treatment in breast cancer, and that FOXC1 may be used as a potential therapeutic target in ERα‐negative breast cancer.

Abstract 3485: Estrogen receptor ligands and their responses in de novo and tamoxifen resistant cell models

Abstract Approximately 75% of breast cancer incidences are Estrogen Receptor positive (ER+). Tamoxifen, a selective estrogen receptor modulator (SERM), is the standard of care for many of these ER+ breast cancer patients. Unfortunately, tamoxifen resistance occurs in almost 50% of patients within 5 years of treatment, and endocrine-independence accompanying resistance negates the effects of aromatase inhibitors. Paradoxically, estradiol (E2) has shown clinical efficacy in patients with resistant breast cancer. Understanding the antiproliferative role of E2 and ER signaling in resistant ER+ cell lines is essential to gain a better understanding of paradoxical clinical efficacy and for the appropriate biomarker-assisted selection of endocrine therapy for various stages of ER+ breast cancer. We have discovered ER ligands, based upon a single chemical scaffold with a diverse set of pharmacological responses, which can be used to better understand the role of ER signaling in resistance and therapy: selective ER modulators (SERMs), selective ER downregulators (SERDs), selective estrogen mimics (SEMs), and selective human ER partial agonists (ShERPAs). These compounds were initially classified using an ERE luciferase reporter assay and affinity for ER confirmed by biochemical assays. The effects of these novel ER-directed chemical probes on cell viability were further examined in multiple 2D and 3D spheroid models of tamoxifen resistance. Finally, ERα localization upon administration of these ligands in tamoxifen-sensitive and tamoxifen-resistant cells was studied. While SERDs and SEMs showed growth inhibition in tamoxifen resistant cell lines, both molecules had different responses and mechanisms of growth inhibition. ERα was localized to extranuclear sites upon administration of E2, SEMs, and ShERPAs, an observation specific to the resistant phenotype and mechanistically associated with spheroid disintegration. SERDs inhibited the antiproliferative actions of E2, but were antiproliferative in resistant cell lines. Further dissection of the role of ER in resistance and survival is needed to define the appropriate ER-directed, endocrine therapy in ER+ breast cancer. Citation Format: Lauren M. Gutgesell, Gregory R. J. Thatcher, Hitisha Patel, Rui Xiong. Estrogen receptor ligands and their responses in de novo and tamoxifen resistant cell models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3485.

Abstract P5-09-16: Contribution of estrone-sulfate to cell proliferation in aromatase inhibitor resistance of estrogen receptor positive breast cancer

Abstract [Background] Estrogen receptor (ER) positive breast cancer was composed of about 70% of clinical breast cancer and therapy for the ER positive breast cancer have progressed, but some patients recur after endocrine therapy including aromatase inhibitors (AIs). The switch of proliferation drive to human epidermal growth factor receptor 2 was reported as one of AI resistance mechanisms, but ER status of many recurrent patients after AI treatment was maintained or slightly decreased. Moreover, plasma concentration of estrone-sulfate (E1S) is mostly unchanged after menopause and treatment of AI. Thus, we established AI resistant model with conserved ER expression and its activity from MCF-7 cell, and found that E1S contributed to proliferation of the AI-resistant cells. [Material and Method] We introduced plasmids carrying ERE-green fluorescent protein (GFP) gene to MCF-7 (E10), and then aromatase gene was additionally introduced to E10 (E10arom). E10arom was cultured in estrogen-depleted medium including testosterone and letrozole, monitoring GFP as ER activity for about three months. We finally established ER positive Letrozole-Resistant cell lines (LR cells). In addition, to confirm whether E1S-dependent growth in LR cells could be reproduced in ER positive breast cancer cell line, we also established human steroid sulfatase (STS) gene-transfected MCF-7 (MCF-STS) and organic anion transporter peptide (OATP) 4A1 gene-transfected MCF-7 (MCF-4A1). ER activity of these cell lines was examined by ERE-luciferase reporter assay, and response to each steroid and drug was investigated by cell proliferation assay. Quantitative PCR was used for comparing expression of mRNA in these cell lines. [Results] mRNA expression of STS and four OATPs including OATP4A1 increased in LR cells comparing to E10arom. Moreover, there was no change in mRNA expressions of efflux drug transporters, aromatase-metaboic enzyme and enzymes related with production for estrogenic androgen. ERE-luciferase assay with E1S showed higher ER activity of LR cells than E10arom, and LR cells were more proliferated by E1S in dose-dependent manner than the parental cell. STS inhibitor alone and its combination with letrozole were inhibitory to proliferation of LR cells. LR cells were also inhibited by selective estrogen receptor modulator (SERM) and downregulator (SERD). In proliferation assay of MCF-STS and MCF-4A1, we observed that these cell lines also proliferated by E1S in dose-dependent manner as LR cells did. [Discussion] Since ER status of recurrent patients after AI treatment was mostly unchanged, our model could be more practical for analysis of AI resistance in ER positive breast cancer. These results showed that combined induction of STS and OATP contributed to estradiol (E2) production from E1S in LR cells, suggesting that analysis of E1S as alternative E2 productive pathway needed investigation of both STS and transporter of E1S. The combination of STS inhibitor with letrozole was effective to LR cells, and SERM and SERD were also effective to these cells, suggesting that combined therapy of STS inhibitor with AI or switching AI to SERM or SERD was beneficial for AI-resistant breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-16.

PD01-07: AR Overexpression and Aromatase Inhibitor Resistance in Breast Cancer.

Abstract Background: Aromatase inhibitors (AIs) have emerged as the therapy of choice for the treatment of estrogen receptor alpha (ERα)-positive breast cancer. However, many patients develop resistance to AI treatment. Although the involvement of the ERα in AI resistance is well established, the role of the androgen receptor (AR) is not known. It has been estimated that about 60%-70% of ERα-positive breast cancer co-express the AR, and that AR agonists can either inhibit or stimulate breast cancer cell proliferation. Thus it is important to determine if there are biomarkers predicting AR's effects in breast tumors. We have previously shown a role for AR-overexpression in tamoxifen resistance in ERα-positive MCF-7 breast cancer cells; here we hypothesized that AR overexpression might similarly be involved in resistance to the AI anastrazole (Anas). Materials and Methods: Stable transfection of MCF-7 cells was performed to generate cell lines that express the aromatase gene (MCF-7 BK Arom) and then co-transfected with an AR expression vector (MCF-7 AR Arom). Aromatase and AR expression levels were evaluated by western blot analysis, and the enzyme activity was evaluated using aromatase activity assays. Proliferation was tested using anchorage independent soft agar assays and MTT in the presence of the androgen substrate androstenedione (AD), or AD plus Anas. ERα and AR transcriptional activities were tested with ERE-luciferase reporter assays. Localization of ERα and AR within the cells was visualized using immunofluorescence microscopy. Results: ERα-positive MCF-7 cells were stably transfected with either aromatase, or aromatase plus AR. MCF-7 aromatase clones overexpressing AR were resistant to the growth inhibitory effects of Anas when stimulated with the androgen AD. Resistance was not mediated through changes in aromatase expression or activity. The growth of several of the AR Arom-overexpressing cells was stimulated with treatment of Anas alone, suggesting that Anas was acting as an agonist. As expected, AD treatment stimulated ERα transcriptional activity, but Anas was unable to block AD-stimulated activity in AR Arom-overexpressing cells using ERE-Luciferase reporter assay. Anas was able to enhance AR and ERα colocalization in AR-overxpressing cells. Resistance was not associated with activation of known mechanisms of resistance, such as HER2, IGF-1R, or MAPK. However AR-overexpressing cells had higher constitutive phosphorylation of Akt. Accordingly, resistance to Anas was blocked using an Akt1/2 inhibitor. Conclusion: Using a model of ERα-positive breast cancer cells expressing exogenous aromatase and AR, we have demonstrated that AR overexpression confers resistance to the AI Anas. These results suggest that in patients recurring on hormonal therapy whose tumors express elevated levels of AR, targeted therapy to Akt might restore hormone sensitivity. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD01-07.

Cloning, expression and functional characterization of carp,Cyprinus carpio, estrogen receptors and their differential activations by estrogens

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in vertebrates and promote the growth and differentiation of the female reproductive system. Importantly, environmental estrogens can influence the reproductive system and have been shown to disrupt gametogenesis in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor ligand interactions in the carp, Cyprinus carpio, a species used widely for both field- and laboratory-based studies, we cloned all three carp estrogen receptors (ER; ERα, ERβ1 and ERβ2) and applied an estrogen-responsive (ERE)-luciferase reporter assay system to characterize the interactions of these receptors with steroidal and synthetic estrogens. DNA fragments encoding all three ERs in carp, ERα, ERβ1 and ERβ2, were obtained from the ovary using degenerate primer sets and PCR techniques, and full-length carp ER (cER) cDNAs were then obtained using RACE (rapid amplification of the cDNA end) techniques. Amino acid sequences of cERs showed overall homology of 46% (α vs β1), 49% (α vs β2) and 53% (β1 vs β2). In the transient transfection ERE-luciferase reporter assay system (using mammalian cells) the cER proteins displayed estrogen-dependent activation of transcription and cERβ2 showed a higher sensitivity to the natural steroid oestrogen, 17β-estradiol, than cERα. The assay system developed is a powerful assay for toxicology and provides a tool for future studies examining the receptor-environmental chemical interactions and estrogen-disrupting mechanisms in carp. The data presented also expand our knowledge of estrogen receptor evolution.

Estrogen receptors in medaka ( Oryzias latipes) and estrogenic environmental contaminants: An in vitro– in vivo correlation

In many vertebrates, estrogens are necessary to promote the growth and differentiation of the female reproductive system during development, and have important reproductive roles in both males and females. Medaka ( Oryzias latipes) has three estrogen receptor (ER) subtypes, ERα, ERβ1 and ERβ2. To evaluate the three medaka ER (mER)-ligand interactions, we applied the ERE-luciferase reporter assay system to characterize each ER subtype. In this transient transfection assay system using mammalian cells, the mER proteins displayed estrogen-dependent activation. 17β-Estradiol (E 2) and op’-DDT showed high activation irrespective of ERs. Endosulfan also exhibited activation; with less/no transactivity measured using other pesticides, i.e., heptachlor, carbendazim, deltamethrin, acephate, dimethoate and amitraz. It was generally observed that ERβ2 had higher activation potential than ERα and ERβ1. To understand the molecular mechanism of estrogen action via ER, we also conducted E 2 treatment where we observed a trigger in ERβ2 expression upon E 2 exposure. The present data suggest that ERβ2 is essential for female gonad maintenance. The data were supported by induction of vitellogenin ( VTG) mRNA in the liver and reduced VTG receptor mRNA expression in the gonad of both sexes. The present work will provide a basic tool allowing future studies to examine the receptor–ligand interactions and endocrine disrupting mechanisms, and also expands our knowledge of estrogen action on reproductive development in medaka.

Cloning and functional characterization of Chondrichthyes, cloudy catshark, Scyliorhinus torazame and whale shark, Rhincodon typus estrogen receptors

Sex-steroid hormones are essential for normal reproductive activity in both sexes in all vertebrates. Estrogens are required for ovarian differentiation during a critical developmental stage and promote the growth and differentiation of the female reproductive system following puberty. Recent studies have shown that environmental estrogens influence the developing reproductive system as well as gametogenesis, especially in males. To understand the molecular mechanisms of estrogen actions and to evaluate estrogen receptor–ligand interactions in Elasmobranchii, we cloned a single estrogen receptor (ESR) from two shark species, the cloudy catshark ( Scyliorhinus torazame) and whale shark ( Rhincodon typus) and used an ERE-luciferase reporter assay system to characterize the interaction of these receptors with steroidal and other environmental estrogens. In the transient transfection ERE-luciferase reporter assay system, both shark ESR proteins displayed estrogen-dependent activation of transcription, and shark ESRs were more sensitive to 17β-estradiol compared with other natural and synthetic estrogens. Further, the environmental chemicals, bisphenol A, nonylphenol, octylphenol and DDT could activate both shark ESRs. The assay system provides a tool for future studies examining the receptor–ligand interactions and estrogen disrupting mechanisms in Elasmobranchii.

2-(4-Hydroxyphenyl)-Benzo[b]thiophen-6-ol, an Estrogen-Like Compound, Induces Apoptosis in T47D/PKCα Breast Cancer Cells.

Abstract Background: Tamoxifen (TAM) treatment failure is a major obstacle encountered in the clinical setting. Our lab has previously shown that constitutive overexpression of PKCα imparts a TAM resistant/ hormone independent phenotype in the T47D:A18 breast cancer cell line. Furthermore 17β-estradiol (E2) inhibits tumor growth in vivo as well as inhibits colony formation when cells are grown in 3D Matrigel (Zhang, 2009). Before TAM treatment was introduced, breast cancer patients received high-dose E2 and diesthystilbesterol (DES) treatment. DES treated patients may have a survival advantage over TAM treated patients in the long term (Peethambaram,1999). Others have shown that E2 can have inhibitory effects on MCF-7 cells both in vitro and in vivo (Lewis-Wambi, 2009). Taken together, this suggests an estrogenic compound may be efficacious for TAM-resistant breast cancer. The present study was designed to evaluate the effect of an estrogenic compound, 2-(4-hydroxyphenyl)-benzo[b]thiophen-6-ol (BTC), related to the SERMs raloxifene and arzoxifene, on the T47D:A18/PKCα breast cancer cell line.Methods: To determine if BTC can activate estrogen regulated genes we preformed an ERE-luciferase reporter assay 24 hours after BTC treatment. 3D Matrigel colony formation assay was performed to determine if BTC could inhibit colony formation. Colonies were stained and counted on day 10. To determine if apoptosis was occurring, cells were grown in Matrigel and the TUNEL Assay was performed on Day 6.Results: T47D:A18/PKCα cells showed a 31-fold induction in ERE-luciferase reporter activity when treated with BTC (10-7M) compared to 7-fold induction when treated with E2. In the T47D:A18/Neo parental cell line, BTC induced a 40-fold induction whereas estradiol induced a 96-fold induction. In the Matrigel colony formation assay T47D:A18/PKCα cells formed significantly fewer colonies when treated with BTC. Conversely, in the T47D:A18/Neo parental cell line BTC enhanced colony formation similar to E2 treatment. Our results indicate that BTC was able to induce significant apoptosis on day 6 (P= 0.038) as determined by the TUNEL Assay. Therefore BTC acts similarly to estradiol by inhibiting T47D:A18/PKCα colony formation and inducing apoptosis when cells are grown in Matrigel.Conclusion: These findings suggest that BTC is a potential lead compound in the treatment of PKCα overexpressing breast cancer. Furthermore, BTC was shown to be a potent inducer of the cytoprotective enzyme NQO1 in the Hepa 1c1c7 cell system as well as a strong activator of antioxidant responsive elements (Yu, 2007) indicating it may have the added benefit of chemoprotective effects which are absent in estradiol treatment. Further understanding of the mechanism in which BTC induces apoptosis in the T47D:A18/PKCα cell line may result in a clinical advantage to estradiol treatment including fewer side effects. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5140.

A single nucleotide polymorphism in steroid receptor coactivator 1 diminishes ERa coactivation.

Abstract Abstract #605 Background: Single nucleotide polymorphisms (SNPs) can contribute to alterations in treatment efficiency. The 3814C>T SNP that causes a P1272S amino acid change located in the activation domain-2 (AD2) of the steroid receptor coactivator SRC-1, has been shown to be associated with decreased bone mineral density in women undergoing tamoxifen treatment for breast cancer. It is well described that members of the SRC family are regulated by posttranslational modifications. Phosphorylation has been shown to be critical for their activity. It is known that histone-acetyl-transferase (HAT) activity of SRC proteins can be controlled through phosphorylation. The C-terminus of SRC-1 is one of the major region to be phosphorylated, possibly to perform proper HAT activity. Therefore, a change from proline to serine at position 1272 is likely to have impact on phosphorylation and subsequent HAT activity. Additionally, in silico analysis of the sequence suggests that the variant removes a potential Glycogen synthase kinase 3 (GSK3) target motif at amino acid 1275. Intriguingly, phosphorylation by GSK3 has been shown to be involved in modulating the activity and functional lifetime of another member of the SRC family, SRC-3.
 Methods: In silico analysis to identify changes in phosphorylation sites after introducing the SNP was performed using http://www.cbs.dtu.dk/services/NetPhos. Mutants P1272S, P1272A, P1272E were generated by mutagenesis using QuickChange® Site-directed Mutagenesis Kit following the manufacturer's protocol. ERE-Luciferase reporter assays were conducted in HEK293 cells to compare the ability of SRC-1 wildtype (WT), variant P1272S, and mutants P1272A or P1272E to activate ERα-mediated transcription. Protein levels were determined after immunoblotting.
 Results: Post-translational modifications, particularly in the form of phosphorylation, are critical for the activity of members from the p160 family of coactivators as described above. Thus, we examined if the introduction of P1272S induces or destroys any phosphorylation sites within SRC-1. We identified phosphorylation sites by scoring the SRC-1 protein sequence including proline (WT) versus serine (SNP). Indeed we found that a cdc2 phosphorylation site is created and a cdk5 phosphorylation site is partially destroyed when serine is present instead of proline. Using an ERE-Luciferase reporter assay in HEK293 cells, we observed a significant loss of co-activation by the variant compared to SRC-1 WT. Transfecting P1272E showed a similar decrease to P1272S in these assays, suggesting the effect of decreased coactivation might be due to the introduction of a new phosphorylation site. Interestingly, transfecting P1272A was not able to rescue the phenotype of P1272S. This finding suggests an additional role for changes in protein folding with subsequent changes in protein activity. Furthermore, immunoblotting of SRC-1 WT or P1272S after estrogen treatment revealed a stabilizing effect of SRC-1 WT but not for the SNP. This further implies that introducing this SNP results in a decreased protein level and subsequently in decreased activity after hormone treatment. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 605.

In vitro assessment of transcriptional activation of the estrogen and androgen receptors of mosquitofish, Gambusia affinis affinis

Sex-steroid hormones are essential for normal reproductive activity in both sexes. Estrogens are necessary for ovarian differentiation during a critical developmental stage in many vertebrates and promote the growth and differentiation of the female reproductive system. Androgens play essential roles in the development and functioning of the vertebrate male reproductive system as well as actively supporting spermatogenesis. Importantly, recent studies suggest that androgens and estrogens have important reproductive roles in both males and females. To understand the molecular mechanisms of estrogen and androgen actions and to evaluate estrogen and androgen receptor–ligand interactions in the mosquitofish, Gambusia affinis affinis, we used degenerate primer sets and PCR techniques to isolated DNA fragments encoding estrogen receptor alpha (ERα; ESR1), ERbeta1 (ERβ1) and ERβ2 from the ovary. Full-length mosquitofish ER (mfER) cDNAs were obtained using cDNA library screening and RACE techniques. Amino acid sequences of mfERs showed over-all homology of 46% (α versus β1), 43% (α versus β2), and 52% (β1 versus β2). We applied the ERE-luciferase reporter assay system to characterize these receptors. In this transient transfection assay system using mammalian cells, the mfER proteins displayed estrogen-dependent activation of transcription. In addition to ERs, the transactivation of mosquitofish ARs (mfARs) previously isolated by our group, were examined using an androgen-responsive MMTV-luciferase assay system. Mosquitofish ARs showed androgen-dependent activation of transcription from the MMTV promoter. These data provide a basic tool allowing future studies examining the receptor–ligand interactions and endocrine disrupting mechanisms in mosquitofish and also expands our knowledge of estrogen and androgen receptor evolution.

Activation of Estrogen Receptor-Mediated Gene Transcription by the Equine Estrogen Metabolite, 4-Methoxyequilenin, in Human Breast Cancer Cells

4-Methoxyequilenin (4-MeOEN) is an O-methylated metabolite in equine estrogen metabolism. O-methylation of catechol estrogens is considered as a protective mechanism; however, comparison of the properties of 4-MeOEN with estradiol (E(2)) in human breast cancer cells showed that 4-MeOEN is a proliferative, estrogenic agent that may contribute to carcinogenesis. 4-MeOEN results from O-methylation of 4-hydroxyequilenin, a major catechol metabolite of the equine estrogens present in hormone replacement therapeutics, which causes DNA damage via quinone formation, raising the possibility of synergistic hormonal and chemical carcinogenesis. 4-MeOEN induced cell proliferation with nanomolar potency and induced estrogen response element (ERE)-mediated gene transcription of an ERE-luciferase reporter and the endogenous estrogen-responsive genes pS2 and TGF-alpha. These estrogenic actions were blocked by the antiestrogen ICI 182,780. In the standard radioligand estrogen receptor (ER) binding assay, 4-MeOEN showed very weak binding. To test for alternate ligand-ER-independent mechanisms, the possibility of aryl hydrocarbon receptor (AhR) binding and ER-AhR cross talk was examined using a xenobiotic response element-luciferase reporter and using AhR small interfering RNA silencing in the ERE-luciferase reporter assay. The results negated the possibility of AhR-mediated estrogenic activity. Comparison of gene transcription time course, ER degradation, and rapid activation of MAPK/ERK in MCF-7 cells demonstrated that the actions of 4-MeOEN mirrored those of E(2) with potency for classical and nonclassical estrogenic pathways bracketing that of E(2). Methylation of 4-OHEN may not represent a detoxification pathway because 4-MeOEN is a full, potent estrogen agonist.

Molecular cloning of estrogen receptor alpha (ERα; ESR1) of the Japanese giant salamander, Andrias japonicus

Estrogens are essential for normal reproductive activity in females and males and for ovarian differentiation during a critical developmental stage in many vertebrates. To understand the molecular mechanisms of estrogen action and to evaluate estrogen receptor ligand interactions in the Japanese giant salamander ( Andrias japonicus), we isolated cDNA encoding the estrogen receptor (ER) from the liver. A full-length Japanese giant salamander ER cDNA (jgsER) was obtained using 5′ and 3′ rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the jgsER showed high identity to the Xenopus ERα (ESR1) (77.7%). We have applied both the conventional ERE-luciferase reporter assay system and the GAL4-transactivation system to characterize this receptor. In two different transient transfection assay systems using mammalian cells, the jgsER protein displayed estrogen-dependent activation of transcription. The GAL4-transactivation system showed about 10-fold greater activity of the estrogen receptor by hormone when compared to the conventional ERE-luciferase reporter assay system. Tissue distribution of ERα mRNA was examined and kidney, ovary and liver exhibited expression. This is the first isolation of an estrogen receptor from a salamander and also is the first functional cDNA obtained from the Japanese giant salamander, an endangered species considered a special natural monument of Japan.

The Growth Inhibitory Activity of theCimicifuga racemosaExtract Ze 450 is Mediated through Estrogen and Progesterone Receptors-Independent Pathways

Despite the wide use of Cimicifuga racemosa (CR) extract to treat symptoms associated with menopause and other gynecological disorders, very little is known about its mechanism of action. Therefore, we studied in this report the antiestrogenic and antiproliferative effect of a new CR ethanolic extract, Ze 450, in a MCF-7 cell clone that does not proliferate in response to 17beta-estradiol (E(2)). Using this cell line, we have found that the extract inhibited cell proliferation and showed antiestrogenic activity using an ERE-luciferase reporter assay. The growth inhibitory activity was different from the antiestrogenic activity since the CR extract also inhibited the growth of the ER-negative human breast cancer cell line T-47D. Also, we evaluated the effects of this CR extract on the transcriptional regulation of genes involved in cell cycle progression in the ER-negative cell lines 293T and T-47D and we found that this extract markedly inhibited the luciferase activity driven by the cyclin D1 promoter and increased the transcriptional activity of the p21 gene promoter. Finally, we observed that our CR extract bound to the progesterone receptor B1 but did not show progestin-like activity in the T-47D cell line. These findings provide new mechanistic insights into the antiproliferative activities of CR in ER-positive and ER-negative tumour cell lines and highlight their potential in the management of climacteric disorders in women with a history of breast cancer.

Terpenoids Found in the Umbelliferae Family Act as Agonists/Antagonists for ERα and ERβ: Differential Transcription Activity between Ferutinine-Liganded ERα and ERβ

Phytoestrogens are assumed to affect the endocrine system of animal species similarly to other man-made endocrine disrupters and to exert their effects through estrogen receptors, specifically ERα and ERβ. However, these molecular mechanisms are not fully understood. In this study, 19 phytochemicals were surveyed for agonist and antagonist activities of ERα and ERβ using an ERE–luciferase reporter assay. The results showed that ferutinine is an agonist for ERα and an agonist/antagonist for ERβ, tschimgine is an agonist for both ERα and ERβ, and tschimganidine is an agonist for only ERα. Ferutinine and tschimganidine are sesquiterpenoids, and tschimgine is a monoterpenoid derived from the Umbelliferae family. A competitive binding assay showed that ferutinine has higher binding affinities than tamoxifen for both ERs. Co-transfections of coactivators such as SRC-1, TIF2, AIB1, and TRAP220 in 293T cells and use of the luciferase assay revealed that TRAP220 failed to enhance the transcription mediated by ERβ in the presence of ferutinine. Moreover, a GST pull-down assay showed that TRAP220 marginally bound to ERβ ligand binding domain in the presence of ferutinine. These results suggest that the conformation of ferutinine-liganded ERβ is difficult for TRAP220 to recognize. Taken together, this suggests that some terpenoids can modulate estrogen signaling as ER subtype-selective phytoestrogens similar to SERMs (selective estrogen receptor modulators).