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E2 suppressed the expression of PPARγ target gene ACOX3 in three breast cancer cell lines. MCF-7 cells were transferred from phenol red containing medium to E2-free medium for 3 days. Next, (A) MCF-7, (B) MCF-7:5C, and (C) MCF-7:2A cells were seeded in six-well plates. After 24 hours, cells were treated with a vehicle control (0.1% EtOH) or E2 (1 nM) for 24, 48, 72 hours. Cells were then harvested in TRIzol reagent. ACOX3 expression levels were quantitated using RT-PCR. *P

(A) The JNK inhibitor effectively blocked phosphorylation of JNK. MCF-7:2A cells were treated with vehicle (0.1% DMSO) or SP600125 (10-5 mol/L) for 24 hours. p-JNK was examined by Western blot. Total JNK was measured as loading control. (B) The JNK inhibitor could not block E2-induced apoptosis in MCF-7:2A cells. MCF-7:2A cells were treated with vehicle (0.1% DMSO; con), E2 (10-9 mol/L), SP600125 (10-5 mol/L), and E2 (10-9 mol/L) plus SP600125 (10-5 mol/L) for 6 days. Annexin V binding assay was used to detect apoptosis. p

Supplementary Figure 2: (A) Reconstructed ion chromatogram for the MRM transition m/z 241 to 208 of standard BTC (100ng/mL); (B) Reconstructed ion chromatogram for the MRM transition m/z 709 to 171 of standard DansylBTC (100ng/ml).

PDF file - 32K, ERalpha and ERbeta mRNA levels in MCF7:5C cells MCF7:5C cells were treated with 0.1% ethanol vehicle (control) and total RNA was isolated after 24h and reverse transcribed and ERalpha and ERbeta mRNA levels was obtained using RT-PCR.

Phytoestrogens have been investigated as natural alternatives to hormone replacement therapy and their potential as chemopreventive agents. We investigated the effects of equol, genistein, and coumestrol on cell growth in fully estrogenized MCF7 cells, simulating the perimenopausal state, and long-term estrogen-deprived MCF7:5C cells, which simulate the postmenopausal state of a woman after years of estrogen deprivation, and compared the effects with that of steroidal estrogens: 17β estradiol (E2) and equilin present in conjugated equine estrogen. Steroidal and phytoestrogens induce proliferation of MCF7 cells at physiologic concentrations but inhibit the growth and induce apoptosis of MCF7:5C cells. Although steroidal and phytoestrogens induce estrogen-responsive genes, their antiproliferative and apoptotic effects are mediated through the estrogen receptor. Knockdown of ERα using siRNA blocks all estrogen-induced apoptosis and growth inhibition. Phytoestrogens induce endoplasmic reticulum stress and inflammatory response stress–related genes in a comparable manner as the steroidal estrogens. Inhibition of inflammation using dexamethasone blocked both steroidal- and phytoestrogen-induced apoptosis and growth inhibition as well as their ability to induce apoptotic genes. Together, this suggests that phytoestrogens can potentially be used as chemopreventive agents in older postmenopausal women but caution should be exercised when used in conjunction with steroidal anti-inflammatory agents due to their antiapoptotic effects. Cancer Prev Res; 7(9); 939–49. ©2014 AACR.

PDF file - 101K, Differential apoptotic effects of E2 equilin and phytoestrogens MCF7:5C cells were treated with control or E2(1nM), equilin (1nM), phytoestrogens (1microM) for 72h and then stained with YO-PRO-1 and propidium iodide and analysed by flow cytometry.

Fulvestrant potentiates the apoptotic effect of salubrinal in MCF7:5C and LCC9 cells.

PDF file - 41K, Anti-estrogen, ICI 182 780 blocks phytoestrogen inhibited growth MCF7:5C cells were treated with E2(1nM), equilin (1nM), phytoestrogens (1microM) in presence of various concentrations of ICI 182 780. Total DNA was assessed in each well using a fluorescent DNA quantification kit.

Approximately 30% of aromatase-inhibitor–resistant, estrogen receptor–positive patients with breast cancer benefit from treatment with estrogen. This enigmatic estrogen action is not well understood and how it occurs remains elusive. Studies indicate that the unfolded protein response and apoptosis pathways play important roles in mediating estrogen-triggered apoptosis. Using MCF7:5C cells, which mimic aromatase inhibitor resistance, and are hypersensitive to estrogen as evident by induction of apoptosis, we define increased global protein translational load as the trigger for estrogen-induced apoptosis. The protein kinase RNA-like endoplasmic reticulum kinase pathway was activated followed by increased phosphorylation of eukaryotic initiation factor-2 alpha (eIF2α). These actions block global protein translation but preferentially allow high expression of specific transcription factors, such as activating transcription factor 4 and C/EBP homologous protein that facilitate apoptosis. Notably, we recapitulated this phenotype of MCF7:5C in two other endocrine therapy–resistant cell lines (MCF7/LCC9 and T47D:A18/4-OHT) by increasing the levels of phospho-eIF2α using salubrinal to pharmacologically inhibit the enzymes responsible for dephosphorylation of eIF2α, GADD34, and CReP. RNAi-mediated ablation of these genes induced apoptosis that used the same signaling as salubrinal treatment. Moreover, combining 4-hydroxy tamoxifen with salubrinal enhanced apoptotic potency.Implications:These results not only elucidate the mechanism of estrogen-induced apoptosis but also identify a drugable target for potential therapeutic intervention that can mimic the beneficial effect of estrogen in some breast cancers.

PDF file - 136K, Diverse effects of E2, equilin and phytoestrogens on cell cycle progression. Distribution of the cells through the cell-cycle phases was analyzed by flow cytometry in cells treated with E2(1nM), equilin (1nM), genistein (1?M), equol (1?M), coumestrol (1?M) or control for 24 h,48h and 72h. The percentage of the cells in each phase is calculated using the ModFit software. The y axis represents the number of cells and FL2-A represents the intensity of propidium iodide.

Supplementary written materials including Supplementary figure/table legends

PDF file - 135K, Chemical structures of estrogens used in the experiments.(A) Steroidal estrogens (B) phytoestrogens.

Perspectives on this Article from Update of the National Surgical Adjuvant Breast and Bowel Project Study of Tamoxifen and Raloxifene (STAR) P-2 Trial: Preventing Breast Cancer

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

Table S5

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. Cancer Prev Res; 4(5); 633–7. ©2011 AACR.

Peroxisome proliferator-activated receptor γ (PPARγ) is an important transcription factor that modulates lipid metabolism and inflammation. However, it remains unclear whether PPARγ is involved in modulation of estrogen (E2)-induced inflammation, thus affecting apoptosis of E2-deprived breast cancer cells, MCF-7:5C and MCF-7:2A. Here, we demonstrated that E2 treatment suppressed the function of PPARγ in both cell lines, although the suppressive effect in MCF-7:2A cells was delayed owing to high PPARγ expression. Activation of PPARγ by a specific agonist, pioglitazone, selectively blocked the induction of TNFα expression by E2, but did not affect other adipose inflammatory genes, such as fatty acid desaturase 1 and IL6. This suppression of TNFα expression by pioglitazone was mainly mediated by transrepression of nuclear factor-κB (NF-κB) DNA-binding activity. A novel finding was that NF-κB functions as an oxidative stress inducer in MCF-7:5C cells but an antioxidant in MCF-7:2A cells. Therefore, the NF-κB inhibitor JSH-23 displayed effects equivalent to those of pioglitazone, with complete inhibition of apoptosis in MCF-7:5C cells, but it increased E2-induced apoptosis in MCF-7:2A cells. Depletion of PPARγ by siRNA or the PPARγ antagonist T0070907 accelerated E2-induced apoptosis, with activation of NF-κB–dependent TNFα and oxidative stress. For the first time, we demonstrated that PPARγ is a growth signal and has potential to modulate NF-κB activity and oxidative stress in E2-deprived breast cancer cell lines. All of these findings suggest that anti-PPARγ therapy is a novel strategy to improve the therapeutic effects of E2-induced apoptosis in E2-deprived breast cancer.

Supplementary Figure 6: Plasma concentration-time profile of BTC following the administration of a single oral dose of 10 mg/Kg BTC to mice. Each data point represents the mean value {plus minus} SD (n = 3).

Supplementary figure/table materials except Table S1 and Table S5

Supplementary Figure 3: Reagents and conditions: (a) N-Bromoacetamide, DCM, EtOH, rt; (b) BBr3, dry DCM, 0sup>; (c) Dansyl chloride, TEA, DCM, 60 sup>C.

Details of Annexin V staining method

Supplementary Figure 4: The calibration standards were prepared by spiking working solution and IS (20ng/mL) into blank mouse plasma, giving final BTC concentrations of 5,10, 20, 30, 40, 50, and 100 ng/mL.

Basal expression levels of stress-related genes in MCF-7:5C and MCF-7:2A cells.

Patients with long-term estrogen-deprived breast cancer, after resistance to tamoxifen or aromatase inhibitors develops, can experience tumor regression when treated with estrogens. Estrogen's antitumor effect is attributed to apoptosis via the estrogen receptor (ER). Estrogen treatment can have unpleasant gynecologic and nongynecologic adverse events; thus, the development of safer estrogenic agents remains a clinical priority. Here, we study synthetic selective estrogen mimics (SEM) BMI-135 and TTC-352, and the naturally occurring estrogen estetrol (E4), which are proposed as safer estrogenic agents compared with 17β-estradiol (E2), for the treatment of endocrine-resistant breast cancer. TTC-352 and E4 are being evaluated in breast cancer clinical trials. Cell viability assays, real-time PCR, immunoblotting, ERE DNA pulldowns, mass spectrometry, X-ray crystallography, docking and molecular dynamic simulations, live cell imaging, and Annexin V staining were conducted in 11 biologically different breast cancer models. Results were compared with the potent full agonist E2, less potent full agonist E4, the benchmark partial agonist triphenylethylene bisphenol (BPTPE), and antagonists 4-hydroxytamoxifen and endoxifen. We report ERα's regulation and coregulators’ binding profiles with SEMs and E4. We describe TTC-352′s pharmacology as a weak full agonist and antitumor molecular mechanisms. This study highlights TTC-352′s benzothiophene scaffold that yields an H-bond with Glu353, which allows Asp351-to-helix 12 (H12) interaction, sealing ERα's ligand-binding domain, recruiting E2-enriched coactivators, and triggering rapid ERα-induced unfolded protein response (UPR) and apoptosis, as the basis of its anticancer properties. BPTPE's phenolic OH yields an H-Bond with Thr347, which disrupts Asp351-to-H12 interaction, delaying UPR and apoptosis and increasing clonal evolution risk.

Supplementary Figure 1: Western blot showing ERalpha expression in T47D:A18/neo, T47D:A18/PKCalpha, T47D:A18, T47D:A18-TAM1, MCF-7:WS8 and MCF-7 5C cell lines.

PDF file - 65K, The sequence of the primers used in the study.

Legends of supplementary Figures

Supplementary Tables from Exemestane's 17-hydroxylated metabolite exerts biological effects as an androgen

Adobe PDF - MCT-07-0312--Suppl_Fig_S2.pdf from Exemestane's 17-hydroxylated metabolite exerts biological effects as an androgen

Aromatase inhibitors (AI) are being evaluated as long-term adjuvant therapies and chemopreventives in breast cancer. However, there are concerns about bone mineral density loss in an estrogen-free environment. Unlike nonsteroidal AIs, the steroidal AI exemestane may exert beneficial effects on bone through its primary metabolite 17-hydroexemestane. We investigated 17-hydroexemestane and observed it bound estrogen receptor α (ERα) very weakly and androgen receptor (AR) strongly. Next, we evaluated 17-hydroexemestane in MCF-7 and T47D breast cancer cells and attributed dependency of its effects on ER or AR using the antiestrogen fulvestrant or the antiandrogen bicalutamide. 17-Hydroexemestane induced proliferation, stimulated cell cycle progression and regulated transcription at high sub-micromolar and micromolar concentrations through ER in both cell lines, but through AR at low nanomolar concentrations selectively in T47D cells. Responses of each cell type to high and low concentrations of the non-aromatizable synthetic androgen R1881 paralleled those of 17-hydroexemestane. 17-Hydroexemestane down-regulated ERα protein levels at high concentrations in a cell type–specific manner similarly as 17β-estradiol, and increased AR protein accumulation at low concentrations in both cell types similarly as R1881. Computer docking indicated that the 17β-OH group of 17-hydroexemestane relative to the 17-keto group of exemestane contributed significantly more intermolecular interaction energy toward binding AR than ERα. Molecular modeling also indicated that 17-hydroexemestane interacted with ERα and AR through selective recognition motifs employed by 17β-estradiol and R1881, respectively. We conclude that 17-hydroexemestane exerts biological effects as an androgen. These results may have important implications for long-term maintenance of patients with AIs. [Mol Cancer Ther 2007;6(11):2817–27]

Supplementary Fig. S1 from Exemestane's 17-hydroxylated metabolite exerts biological effects as an androgen

Supplementary Figure Legends from Exemestane's 17-hydroxylated metabolite exerts biological effects as an androgen

Supplementary Figure 4. MCF-7:WS8 cells were treated with vehicle, 1nM E2, or 10nM, 100nM or 1µM concentrations of Dex, MPA, or NETA for 24 hours. PR mRNA expression was quantified by RT-PCR. 36B4 was used as an internal control. Means represent three samples in triplicate.

PDF file - 53K, 7A, Inhibitors of Akt could not block growth inhibition induced by E2 in MCF-7:5C cells. MCF-7:5C cells were treated with vehicle (0.1% EtOH), E2 (10-9 mol/L), LY294002 (10-6mol/L), and E2 (10-9 mol/L) plus LY294002 (10-6mol/L) respectively. Cells were harvested after 7 days treatment and total DNA was determined using a DNA fluorescence quantitation kit. 7B, Inhibitor of MAPK could not block growth inhibition induced by E2 in MCF-7:5C cells. MCF-7:5C cells were treated with vehicle (0.1% EtOH), E2 (10-9 mol/L), U0126 (5x10-6 mol/L), and E2 (10-9 mol/L) plus U0126 (5x10-6 mol/L) respectively. Cells were harvested after 7 days treatment and total DNA was determined using a DNA fluorescence quantitation kit. All data shown were representative of at least three separate experiments with similar results.

PDF file - 82K, 6A, The c-Src inhibitor blocked tumor necrosis factor (TNF) super family genes induced by E2. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT (10-6 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 72 hours. Cells were harvested in TRIzol. LTA (TNF super family member 1) gene was detected by real-time PCR. P

Supplementary Figure Legends. Supplementary Figure Legends

PDF file - 77K, 1A, The c-Src inhibitor blocked E2 activated non-genomic pathway. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 10 minutes and the cell lysates were harvested. Phosphorylated MAPK and c-Src were examined by immunoblotting with primary antibodies. Immunoblotting for total MAPK and c-Src were used for loading controls. 1B, E2 rapidly activated MAPK and c-Src. MCF-7:5C cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cell lysates were harvested. Phosphorylated MAPK and c-Src were examined by immunoblotting with primary antibodies. Immunoblotting for total MAPK and c-Src were used for loading controls. 1C, E2 stimulated c-Src after 24 hours treatment. MCF-7:5C cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cell lysates were harvested. Phosphorylated c-Src was examined by immunoblotting with primary antibody. Immunoblotting for total c-Src was used for loading control. 1D, Quantification of Annexin V binding assay. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT (10-6 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 72 hours and the cells were harvested for Annexin V binding assay through flow cytometry. The percentage of Annexin V binding was compared with control. P

Supplementary Figure 2. MCF-7:5C cells were treated with Dex (A), MPA (B), or NETA (C) at concentrations ranging from 10-10 M to 10-6 M for seven days, and DNA was quantified.

Supplementary Figure 1 from The G Protein–Coupled Receptor GPR30 Inhibits Proliferation of Estrogen Receptor–Positive Breast Cancer Cells

PDF file - 47K, 5A, The oxidative stress indicator HMOX1 expressed in wild-type MCF-7 cells after E2 treatment. Wild-type MCF-7 cells were cultured in E2 free medium for three days. Then, cells were plated in six-well plates. After one day, cells were treated with vehicle (0.1% EtOH) and E2 (10-9 mol/L) for different time points as indicated and the cells were harvested in TRIzol for real-time PCR. P

PDF file - 76K, The c-Src inhibitor blocked apoptosis-related genes induced by E2. MCF-7:5C cells were treated with vehicle (0.1% DMSO), E2 (10-9 mol/L), 4-OHT (10-6 mol/L), E2 (10-9 mol/L) plus 4-OHT(10-6 mol/L), PP2 (5x10-6 mol/L), E2 (10-9 mol/L) plus PP2 (5x10-6 mol/L) respectively for 72 hours. Cells were harvested in TRIzol for real-time PCR. 4A, PPP1R15A (GADD34) gene. 4B, BCL2L11 (Bim) gene. 4C, NUAK2 gene. 4D, PMAIP1 (Noxa) gene. P

Supplementary Figure 1. A. MCF-7:WS8 and MCF-7:5C cells were treated with either vehicle for 72 hours or 1nM E2 for 24 or 72 hours. Cells were then harvested, and ERα, PR, and GR proteins were measured by Western blot. MCF-7:WS8 cells were used as a positive control for PR expression. B. MCF-7:5C cells were treated with vehicle, 1nM E2, 1µM Dex, MPA, or NETA, or combinations of 1nM E2 plus 1µM Dex, MPA, or NETA for two months. Cells were harvested, proteins were extracted, and Western blots for ERα and GR protein levels were performed. β-actin was used as a loading control.

Supplementary Figure 6. A. MCF-7:WS8 cells were transfected with an ERE-luciferase reporter construct for 24 hours, then treated with vehicle, 1µM ICI, 1µM R5020, 1µM RU486, 1nM E2, 1µM MPA, 1µM NETA, or combinations for 24 hours. ERE activity was measured by luciferase assay and normalized to vehicle control. Means represent samples in triplicate. B. MCF-7:WS8 cells were transfected with an ERE-luciferase reporter construct for 24 hours, then treated with vehicle, 1µM 4-OHT, 1µM R5020, 1µM RU486, 1nM E2, 1µM MPA, 1µM NETA, or combinations for 24 hours. ERE activity was measured by luciferase assay and normalized to vehicle control. Means represent samples in triplicate. Error bars too small to visualize.

Supplementary Methods from The G Protein–Coupled Receptor GPR30 Inhibits Proliferation of Estrogen Receptor–Positive Breast Cancer Cells

Supplementary Figure 5. MCF-7:5C cells were transfected with an ERE-luciferase reporter construct for 24 hours, then treated with vehicle, 1µM 4-OHT, 1nM E2, 1µM MPA, 1µM NETA, or combinations for 24 hours. ERE activity was measured by luciferase assay and normalized to vehicle control. Means represent samples in triplicate. Error bars too small to visualize.