Puerarin Suppresses Proliferation of Endometriotic Stromal Cells Partly via the MAPK Signaling Pathway Induced by 17ß-estradiol-BSA

<div><h3>Background</h3><p>Puerarin is a major isoflavonoid compound extracted from <em>Radix puerariae</em>. It has a weak estrogenic action by binding to estrogen receptors (ERs). In our early clinical practice to treat endometriosis, a better therapeutic effect was achieved if the formula of traditional Chinese medicine included <em>Radix puerariae</em>. The genomic and non-genomic effects of puerarin were studied in our Lab. This study aims to investigate the ability of puerarin to bind competitively to ERs in human endometriotic stromal cells (ESCs), determine whether and how puerarin may influence phosphorylation of the non-genomic signaling pathway induced by 17ß-estradiol conjugated to BSA (E₂-BSA).</p> <h3>Methodology</h3><p>ESCs were successfully established. Binding of puerarin to ERs was assessed by a radioactive competitive binding assay in ESCs. Activation of the signaling pathway was screened by human phospho-kinase array, and was further confirmed by western blot. Cell proliferation was analyzed according to the protocol of CCK-8. The mRNA and protein levels of cyclin D1, Cox-2 and Cyp19 were determined by real-time PCR and western blotting. Inhibitor of MEK1/2 or ER antagonist was used to confirm the involved signal pathway.</p> <h3>Principal Findings</h3><p>Our data demonstrated that the total binding ability of puerarin to ERs on viable cells is around 1/3 that of 17ß-estradiol (E₂). E₂-BSA was able to trigger a rapid, non-genomic, membrane-mediated activation of ERK1/2 in ESCs and this phenomenon was associated with an increased proliferation of ESCs. Treating ESCs with puerarin abrogated the phosphorylation of ERK and significantly decreased cell proliferation, as well as related gene expression levels enhanced by E₂-BSA.</p> <h3>Conclusions/Significance</h3><p>Puerarin suppresses proliferation of ESCs induced by E₂-BSA partly via impeding a rapid, non-genomic, membrane-initiated ERK pathway, and down-regulation of Cyclin D1, Cox-2 and Cyp19 are involved in the process. Our data further show that puerarin may be a new candidate to treat endometriosis.</p> </div

Phospho-proteomic profiling of E₂-BSA and puerarin effects on ERK1/2 phosphorylation in ESCs and confirmed by western blot analysis.

<p>Cells were treated as indicated. A. AKT, Chk2 and ERK1/2 were activated by E₂-BSA and suppressed by puerarin. B. Western blot analysis of E₂-BSA on phosphorylation in ESCs. Cells were treated with vehicle (DMSO) or E₂-BSA (10⁻⁸ mol/L) for 15 min. AKT, Chk2 and ERK1/2 were activated by E₂-BSA. Expression was normalized to total ERK1/2.</p

Analysis of expression of cyclin D1, cyp19 and cox-2 genes in ESCs subjected to various treatments.

<p>ESCs were treated with E₂-BSA, puerarin or both for 24 h, in the absence and presence of pretreatment with U0126 for 60 min. Total RNA was isolated, RT quantitative real-time PCR was performed and relative expression of cyclin D1 (A), cyp19 (B) and cox-2 (C) were obtained. GAPDH served as an internal control. Individual bars show relative mRNA levels expressed as the mean ± S.E.M., determined from three separate assays. Protein levels were detected by western blotting (D). Group <i>vs.</i> control, **, <i>P</i><<i>0.01 and</i> *, <i>P</i><<i>0.05;</i> Group <i>vs.</i> E₂-BSA, <i>▴▴, P</i><<i>0.01</i> and <i>▴, P</i><<i>0.05</i>.</p

Estrogen receptor competitive binding assay.

<p>ESCs were incubated in the presence of E₂ or puerarin solutions for 1 h. After removal of the medium, [³H]-E₂ binding capacity was measured. (A) Total binding of [³H]-E₂ to ERs in ESCs; the concentrations of [³H]-E₂ used were 0, 0.1950, 0.3906, 0.7813, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 nmol/L. (B) Non-specific binding of [³H]-E₂. (C) Specific binding of [³H]-E₂, calculated by subtracting the non-specific binding values from the total binding values. (D) Measurement of [³H]-E₂ binding to ERs in the presence of varying concentrations of E₂ (0, 0.0061, 0.0183, 0.0549, 0.1646, 0.4938, 1.4815, 4.4444, 13.3333 and 40 µmol/L) or puerarin (0, 0.0061, 0.0183, 0.0549, 0.1646, 0.4938, 1.4815, 4.4444, 13.3333 and 40 µmol/L). The figures represent data from three experiments; each experiment was performed in triplicate, and data are represented as the mean ± SD.</p

Effect of E₂-BSA and puerarin on cell proliferation in ESCs.

<p>(A) ESCs were treated with various concentrations (10⁻¹⁰ to 10⁻⁶ mol/L) of E₂-BSA for 4 d. (B) Growth curves of ESCs incubated with E₂-BSA (10⁻⁸ mol/L) and/or puerarin (10⁻⁹ mol/L) for 0, 1, 2, 4 d. (C) Cells were treated for 4 d with E₂-BSA (10⁻⁸ mol/L) in the absence or presence of puerarin (10⁻⁹ mol/L) and/or pretreated with U0126 (20 µmol/L) for 60 min. The figures represent data obtained from three experiments; each experiment was performed in triplicate, and data are represented as the mean ± SD. DMSO treatment was used as the vehicle control. <i>Vs. control</i>,**, <i>P</i><<i>0.01,</i> *, <i>P</i><<i>0.05; vs. E₂-BSA, ▴, P</i><<i>0.05.</i></p