Liquiritigenin is a plant-derived highly selective estrogen receptor beta agonist

Abstract

After the Women's Health Initiative found that the risks of hormone therapy outweighed the benefits, a need for alternative drugs to treat menopausal symptoms has emerged. We explored the possibility that botanical agents used in Traditional Chinese Medicine for menopausal symptoms contain ERbeta-selective estrogens. We previously reported that an extract containing 22 herbs, MF101 has ERbeta-selective properties. In this study we isolated liquiritigenin, the most active estrogenic compound from the root of Glycyrrhizae uralensis Fisch, which is one of the plants found in MF101. Liquiritigenin activated multiple ER regulatory elements and native target genes with ERbeta but not ERalpha. The ERbeta-selectivity of liquiritigenin was due to the selective recruitment of the coactivator steroid receptor coactivator-2 to target genes. In a mouse xenograph model, liquiritigenin did not stimulate uterine size or tumorigenesis of MCF-7 breast cancer cells. Our results demonstrate that some plants contain highly selective estrogens for ERbeta.

Fig. 1

Structure of liquiritigenin. The active compound was isolated from dry, powdered G. uralensis and identified by NMR.

Fig. 2

Liquiritigenin selectively activates transcription through ERβ. (A) ERE tk-Luc was cotransfected into U2OS cells with expression vectors for ERα or ERβ. After transfection, the cells were treated for 18 h with liquiritigenin and luciferase activity was measured. Dose-response curves for E₂ and liquiritigenin in U2OS cells. Following transfection the cells were treated with increasing amounts of E₂ or liquiritigenin for 18 h (B). ERE tk-Luc was cotransfected into U2OS cells with expression vectors for ERα (left panel) or ERβ (right panel) and then treated with 10 nM E₂, 1 μM liquiritigenin, 1 μM DPN or 1 μM PPT for 18 h and then luciferase was measured (C). The activation by liquiritigenin is blocked by anti-estrogens. (D) ERE tk-Luc was cotransfected into U2OS cells with an expression vector ERβ and the cells were treated with 1 μM liquiritigenin in the absence or presence of 1 μM ICI 182780 (ICI), raloxifene (Ral) or tamoxifen (Tam). Liquiritigenin selectively activated the ERE tk-Luc with ERβ in HeLa cervical (E) and WAR5 prostate cancer (F) cell lines. Each data point is the average of triplicate determinations ± S.E.M. An activation by the drug was significant (p < 0.05) when it was 2-fold greater than the control values.

Fig. 3

The activation by liquiritigenin is selective for the estrogen receptor. U2OS were transfected with TAT3-luciferase and androgen receptor (AR) (A), MMTV-luciferase and glucocorticoid receptor (GR) (B), TAT3-luciferase and progesterone receptor B (PR) (C), or F2 tk-Luc and thyroid hormone receptor β1 (TR) (D). The cells were treated for 18 h with 1 nM dihydrotestosterone (DHT), or 1 nM dexamethasone (Dex), or 1 nM progesterone (Prog), or 10 nM triiodothyronine (T₃) (A, B, C, D, respectively) or 2.5 μM liquiritigenin (Liq). Each data point is the average of triplicate determinations ± S.E.M. An activation by the drug was significant (p < 0.05) when it was 2-fold greater than the control values.

Fig. 4

Liquiritigenin selectively activates transcription of the native ER regulatory elements and genes through ERβ. CECR6 tk-Luc (A), NKG2E tk-Luc (B), and NKD tk-Luc (C) were cotransfected into U2OS cells with expression vectors for human ERα or ERβ. After transfection, the cells were treated for 18 h with increasing amounts of liquiritigenin and luciferase activity was measured. U2OS cells stably transfected with tetracycline inducible ERβ or ERα were treated with 1 μg/ml doxycycline for 18 h to induce ER expression. The cells were then treated for increasing times with liquiritigenin. The level of CECR6 (D), NKG2E (E), and NKD (F) mRNA was measured by real-time PCR. Each data point is the average of triplicate determinations ± S.E.M. An activation by the drug was significant (p < 0.05) when it was 2-fold greater than the control values.

Fig. 5

Liquiritigenin selectively recruits SRC-2 to the ER target genes. (A) Purified ERα or ERβ were incubated with fluorescent E₂ in the absence or presence of increasing amounts of liquiritigenin. U2OS-ERβ or U2OS-ERα cells were treated with liquiritigenin for increasing times and ChIP was performed using antibodies to SRC-2. Real-time PCR was performed to amplify the ER regulatory element in the CECR6 (B), NKG2E (C), and NKD (D) genes.

Fig. 6

Liquiritigenin does not stimulate tumor formation of MCF-7 breast cancer cells or cause uterine growth in a mouse xenograft model. MCF-7 cells were grafted under the kidney capsule of intact female nude mice. Mice were continuously infused with vehicle (Control), E₂ (0.4 mg) or liquiritigenin (2 mg) using a subcutaneous osmotic pump. After one month, the tumors and uterus were removed and analyzed for size and weight. Gross morphology of the xenografts in control (A), E₂ (B) and liquiritigenin (C) treated mice. The arrow points to the site of grafting. Average weights ± S.E.M of tumor grafts (D) and uterine horns (E) from 5 mice in each group. * indicates a p<0.05 difference between control and drug treatment using the student’s t-test.