Diarylheptanoid phytoestrogens isolated from the medicinal plant Curcuma comosa: biologic actions in vitro and in vivo indicate estrogen receptor-dependent mechanisms

Abstract

Background: Diarylheptanoids isolated from Curcuma comosa Roxb. have been recently identified as phyto estrogens. However, the mechanism underlying their actions has not yet been identified.

Objectives: We characterized the estrogenic activity of three active naturally occurring diarylheptanoids both in vitro and in vivo.

Methods: We characterized mechanisms of estrogenic action of the diarylheptanoids (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol (D1), 1,7-diphenyl-(6E)-6-hepten-3-one (D2), and (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (D3) by using a real-time polymerase chain reaction assay, a mammalian transfection model, and a uterotrophic assay in mice.

Results: All diarylheptanoids up-regulated estrogen-responsive genes in estrogen-responsive breast cancer cells (MCF-7). In HepG2 cells transfected with estrogen receptor (ER) beta or different ERalpha functional receptor mutants and the Vit-ERE-TATA-Luc reporter gene, all diarylheptanoids induced transcription through a ligand-dependent human ERalpha-ERE-driven pathway, which was abolished with ICI 182,780 (ER antagonist), whereas only D2 was active with ERbeta. An ERalpha mutant lacking the functional AF2 (activation function 2) region was not responsive to 17beta-estradiol (E(2)) or to any of the diarylheptanoids, whereas ERalpha lacking the AF1 domain exhibited wild-type-like activity. D3 markedly increased uterine weight and proliferation of the uterine epithelium in ovariectomized mice, whereas D1 and D2 were inactive. D3, like E(2), up-regulated lactoferrin (Ltf) gene expression. The responses to D3 in the uterus were inhibited by ICI 182,780. In addition, D3 stimulated both classical (Aqp5) and nonclassical (Cdkn1a) ER-mediated gene regulation.

Conclusions: The results suggest that the D3 diarylheptanoid is an agonist for ER both in vitro and in vivo, and its biological action is ERalpha selective, specifically requiring AF2 function, and involves direct binding via ER as well as ERE-independent gene regulation.

Keywords: Curcuma comosa; ER dependent; ERE dependent; ERE independent; diarylheptanoid; estrogen-regulated genes; uterotrophic activity.

Figure 1

Structure of the diarylheptanoids (3S)-1,7-diphenyl-(6E)-6-hepten-3-ol (D1), 1,7-diphenyl-(6E)-6-hepten-3-one (D2), and (3R- ) 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (D3), purified compounds isolated from rhizome (Suksamrarn et al. 2008).

Figure 2

Endogenous gene expression in MCF-7 cells treated with EtOH (vehicle), 1 nM E₂, or 1, 10, 20, or 50 μM diarylheptanoid (D1, D2, or D3) in the presence or absence of 1 μM ICI for 24 hr. Fold activation of TFF1 (A), PGR (B), MYC (C), and CTSD (D) compared with EtOH. Each value represents the average increase above EtOH obtained from three independent experiments performed in duplicate. ^*p < 0.05, ^**p < 0.01, and ^#p < 0.001, compared with EtOH.

Figure 3

Relative luciferase activity in HepG2 cells transfected with hERα (A), hERβ (B), and 3 ×-Vit-ERE-TATA-Luc plasmids that we treated with EtOH (vehicle), 1 nM E₂, or 1, 10, 20, 50 μM diarylheptanoid (D1, D2, or D3) in the presence or absence of 1 μM ICI. Each value was obtained from three independent experiments performed in triplicate. ^*p < 0.05, ^**p < 0.01, and ^#p < 0.001, compared with EtOH.

Figure 4

Relative luciferase activity in HepG2 cells transfected with mERαWT (WT mouse ERα), mERKOE1 (deleted AF1 region of mouse ERα), mAF2ER (mutated AF2 region of mouse ERα), and 3×-Vit-ERE-TATA-Luc plasmids that were treated with EtOH (vehicle), 1 nM E₂, 1 μM ICI, or 50 μM D1, D2, or D3. Each value was obtained from three independent experiments performed in triplicate. ^*p < 0.05, compared with EtOH for mERαWT group. ^#p < 0.05, compared with EtOH for mERKOE1 group. ^##p < 0.05, compared with EtOH for mAF2ER group.

Figure 5

Uterine wet weight (mg/100 g body weight (BW); A] and uterine Ltf expression (B) in adult OVX mice treated for 3 consecutive days with sesame oil (vehicle), 0.25 μg E₂, or 2.5 mg diarylheptanoids (D1, D2, or D3). In addition, 45 μg ICI (ER antagonist) or 2 mg flutamide (AR antagonist) were coadministered with 2.5 mg D3. ^*p < 0.05, and ^#p < 0.001, compared with vehicle ( n = 3–4 animals/group).

Figure 6

Ki67 immunohistochemistry. Cross-sectional uterine tissues from adult OVX mice treated 3 consecutive days with sesame oil (vehicle; A), 0.25 μg E₂( B), 45 μg ICI + E₂( C), 2.5 mg D1( D), 2.5 mg D2 (E), 2.5 mg D3 (F), 45 μg ICI + 2.5 mg D3 (G), and 2 mg flutamide + 2.5 mg D3 (H). Tissues are stained with an antibody to the proliferative marker Ki67. Bar = 40 μm.

Figure 7

Effect of D3 on the classical (ERE-mediated) and nonclassical genes in mouse uteri. Aqp5 (A), and Cdkn1a (B) in mouse uteri from adult WT, αERKO, and KI/KO OVX mice treated for 2 hr with vehicle (sesame oil), 0.25 μg E₂, or 2.5 mg D3. Data represent the mean ± SE increase above the vehicle group, with expression ratios normalized to Rpl7 by real-time reverse transcriptase PCR (n = 3–7 animals/group).