Estrogenic Activity of 4-Hydroxy-Benzoic Acid from Acer tegmentosum via Estrogen Receptor α-Dependent Signaling Pathways

Abstract

Acer tegmentosum, a deciduous tree belonging to Aceraceae, has been used in traditional oriental medicine for treating hepatic disorders, such as hepatitis, cirrhosis, and liver cancer. We evaluated the estrogen-like effects of A. tegmentosum using an estrogen receptor (ER)-positive breast cancer cell line, namely MCF-7, to identify potential phytoestrogens and found that an aqueous extract of A. tegmentosum promoted cell proliferation in MCF-7 cells. Five phenolic compounds (1-5) were separated and identified from the active fraction using bioassay-guided fractionation of crude A. tegmentosum extract and phytochemical analysis. The chemical structures of the compounds were characterized as vanillic acid (1), 4-hydroxy-benzoic acid (2), syringic acid (3), isoscopoletin (4), and (E)-ferulic acid (5) based on the analysis of their nuclear magnetic resonance spectra and liquid chromatography-mass spectrometry data. All five compounds were evaluated using an E-screen assay for their estrogen-like effects on MCF-7 cells. Among the tested compounds, only 4-hydroxy-benzoic acid (2) promoted the proliferation of MCF-7 cells, which was mitigated by the ER antagonist, ICI 182,780. The mechanism underlying the estrogen-like effect of 4-hydroxy-benzoic acid (2) was evaluated via western blotting analysis to determine the expression levels of extracellular signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K), serine/threonine kinase (AKT), and ERα. Our results demonstrated that 4-hydroxy-benzoic acid (2) induced the increase in the protein expression levels of p-ERK, p-AKT, p-PI3K, and p-Erα, concentration dependently. Collectively, these experimental results suggest that 4-hydroxy-benzoic acid (2) is responsible for the estrogen-like effects of A. tegmentosum and may potentially aid in the control of estrogenic effects during menopause.

Keywords: 4-hydroxy-benzoic acid; Acer tegmentosum; estrogen receptor; phytoestrogens.

Figure 1

Comparison of the estrogenic activity of the (A) crude extract (total); (B) hexane (Hx), (C) CH₂Cl₂ (MC), (D) EtOAc (EA), and (E) n-BuOH (BuOH) fractions; and (F) 17β-estradiol in the absence or presence of ICI 182,780 according to the cell proliferation measured by E-screen assay in MCF-7 cells. * Significant difference between the cells treated with the extract or fractions and untreated cells. # Significant difference between the cells treated with ICI and without ICI at the same concentration of sample (n = 3 independent experiments, p < 0.05, Kruskal–Wallis non-parametric test). Data are represented as the mean ± standard error of the mean (SEM).

Figure 2

Isolation scheme of compounds 1–5 from the extract of Acer tegmentosum.

Figure 3

Chemical structures of compounds 1–6: vanillic acid (1), 4-hydroxy-benzoic acid (2), syringic acid (3), isoscopoletin (4), and (E)-ferulic acid (5).

Figure 4

Estrogenic effects of the isolated compounds (1–5) in the presence or absence of ICI 182,780 measured by the E-screen assay on MCF-7 cells. Cell viability is expressed as the mean ± SEM. *** p < 0.001 compared to the non-treated group; ^# p < 0.05 compared to the ICI-treated group at the same concentration of samples. Data are represented as the mean ± SEM.

Figure 5

(A) Effects of 4-hydroxy-benzoic acid (2) on the protein expression levels of p-ERK, ERK, p-PI3K, PI3K, p-AKT, AKT, p-ERα, ERα, and GAPDH in MCF-7 cells treated or untreated with 25, 50, and 100 µM 4-hydroxy-benzoic acid (2) for 96 h. (B–E) The bar graph presents the densitometric quantification of the western blot bands. * Significant difference between the treated cells with 4-hydroxy-benzoic acid (2) and untreated cells. Significant difference between the treated cells with ICI and without ICI at the same concentration of the sample (n = 3 independent experiments, p < 0.05, Kruskal–Wallis non-parametric test) (n = 3 independent experiments, p < 0.05, Kruskal–Wallis non-parametric test). Data are represented as the mean ± SEM.

Figure 6

Schematic illustration of the mechanism underlying the estrogenic activity regarding to 4-hydroxy-benzoic acid via ERα-dependent signaling pathways in the ER-positive breast cancer cells (MCF-7).