Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells

Abstract

Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L.) which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα) antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box). In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells). Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif.

Figure 1

Molecular structure of THIAA.

Figure 2

Effect of commercial hop extracts on proliferation (a) and metabolic activity (b) of MCF-7 and MDA-MB-231 cells in serum-complete condition. Cells were grown during 72 h in the absence (control) or presence of indicated hop extracts at 0.001 and 0.01%. Cell proliferation was measured by crystal violet staining and metabolic activity by the MTT method. Measurements were performed in sixplicate. Data refer to the mean value ± SD of a representative experiment performed independently three times.

Figure 3

THIAA induced apoptosis in serum-complete condition. MCF-7 and MDA-MB-231 cells were treated (or not: control) during 24 h with indicated concentrations of THIAA. Early and late apoptosis as well as necrosis were assessed by flow cytometry after annexin V-FITC/propidium iodine labeling. Data refer to the mean value ± SD of three independent experiments; ^∗ p < 0.2; ^∗∗ p < 0.05.

Figure 4

Effect of THIAA on E₂-stimulated MCF-7 cell proliferation in steroid-free culture medium. MCF-7 cells were grown during 72 h in the absence (control) or presence of E₂ at 10⁻⁹M with or without THIAA at indicated concentrations. Cell proliferation was measured by crystal violet staining. Data refer to the mean value ± SD of a representative experiment performed independently three times in sixplicate.

Figure 5

Effect of THIAA on E₂-stimulated cell cycle progression in steroid-free culture medium. MCF-7 cells were grown during 72 h in the absence (control) or presence of E₂ at 10⁻⁹M with or without THIAA at 0.01%. Cell cycle phases were analyzed by flow cytometry after propidium iodine labeling. Data refer to a representative experiment performed independently three times.

Figure 6

Effect of THIAA on E₂-stimulated MCF-7 cell clonogenicity in steroid-free culture medium. Cells were seeded in Petri dishes at low concentration. Cells were grown during 72 h in the absence (control) or presence of E₂ at 10⁻⁹M with or without antiestrogens at 10⁻⁶M or THIAA at 0.01%. Number of colonies (a) and number of cells per colony (b) were counted. For the later, data were subdivided into 4 classes (1-2, 3–6, 7–11, and more than 11 cells) according to a quartile calculation.

Figure 7

Effect of THIAA on ERα-dependent transcription by reporter gene assay in steroid-free culture medium. MVLN cells were incubated for 24 h in the absence (control) or presence of E₂ at 10⁻⁹M with or without THIAA at 0.001%. Luciferase activity was assayed in cellular extracts by luminometry and emitted light signals were expressed in arbitrary units (relative luciferase units (RLU)) per mg protein. Data refer to the mean value ± SD of an experiment performed in triplicate and are representative of three independent experiments.

Figure 8

Effect of THIAA on PgR expression in steroid-free culture medium by western blot. MCF-7 cells were incubated for 24 h in the absence (control) or presence of E₂ at 10⁻⁹M with or without THIAA at 0.001%. Immunoblots and optical density (OD) analyses of PgRA and PgRB refer to an experiment performed independently twice. ODs of both PgR isoforms are normalized by ODs of actin and expressed as percentage of control.

Figure 9

Effect of THIAA on ERα protein level in steroid-free culture medium by immunofluorescence (a) and western blot (b). MCF-7 cells were incubated for 24 h in the absence (control) or presence of E₂ at 10⁻⁹M with or without THIAA at 0.001%. Immunofluorescence, immunoblots, and optical density (OD) analyses of ERα refer to an experiment performed independently twice. ODs of ERα are normalized by ODs of actin and expressed as percentage of control.

Figure 10

Effect of THIAA on [³H]E₂-ERα complexation in MCF-7 cells. MCF-7 cells were incubated during 40 minutes with 10⁻⁹M [³H]E₂ and increasing concentrations of THIAA (a) or E₂ (b). Whole cell [³H]E₂ binding capacity was assessed by liquid scintillation counting. Data (mean ± SD) refer to a representative experiment performed three times in triplicate.

Figure 11

Effect of THIAA on LxxLL motif recruitment assessed by TR-FRET (a) and ELISA (b). For TR-FRET experiments, recombinant ERα was incubated in the absence (control) or presence of E₂ at 10⁻⁶M with or without THIAA at 0.001% or an LxxLL competitor peptide at 10⁻⁴M. Fluorescence emission was measured at 488 nm and 518 nm under a 332 nm excitation wavelength. Data refer to the mean value ± SD of Relative Fluorescence Unit (RLU) ratio at 518 nm and 488 nm of an experiment performed twice in triplicate. For ELISA, cell extracts of MCF-7 treated or not with E₂ at 10⁻⁹M (30 min) were incubated with or without THIAA at 0.001% or an LxxLL competitor peptide at 10⁻⁴M. Cell extracts where then submitted to this ELISA in which the capture element is an LxxLL peptide. Data refer to an experiment performed three times.