Activation of Estrogen Receptor by Bavachin from Psoralea corylifolia

Abstract

In this study, we examined the estrogenic activity of bavachin, a component of Psoralea corylifolia that has been used as a traditional medicine in Asia. Bavachin was purified from ethanolic extract of Psoralea corylifolia and characterized its estrogenic activity by ligand binding, reporter gene activation, and endogenous estrogen receptor (ER) target gene regulation. Bavachin showed ER ligand binding activity in competitive displacement of [(3)H] E2 from recombinant ER. The estrogenic activity of bavachin was characterized in a transient transfection system using ERα or ERβ and estrogen-responsive luciferase plasmids in CV-1 cells with an EC50 of 320 nM and 680 nM, respectively. Bavachin increased the mRNA levels of estrogen-responsive genes such as pS2 and PR, and decreased the protein level of ERα by proteasomal pathway. However, bavachin failed to activate the androgen receptor in CV-1 cells transiently transfected with the corresponding receptor and hormone responsive reporter plasmid. These data indicate that bavachin acts as a weak phytoestrogen by binding and activating the ER.

Keywords: Bavachin; Estrogen receptor; Phytoestrogen; Psoralea corylifolia.

Fig. 1.. The chemical structure of bavachin isolated from Psoralea coryfolia.

Fig. 2.. Competitive inhibition of [³H] E2 binding to hERα (A) and hERβ (B) by bavachin. The hERα and hERβ are incubated with 5 nM [³H]-E2 and test compounds for 2 h and the receptor-bound radioactivities were determined by scintillation counting. The relative specific binding activity (RBA) was calculated as described in experimental section and the IC₅₀ values were determined from the concentration-binding curve (n=4).

Fig. 3.. Dose-dependent ERE-luciferase reporer activity by bavachin in CV-1 cells. Cells were transiently transfected with ERE-luciferase reporter plasmid and either hERα (A) or hERβ (B) expression plasmid. Cells were treated with E2 or bavachin for 24h followed by transient transfection. Cell lysates were prepared and analyzed by luciferase assays. The data are representative of at least three independent experiments performed in triplicate and presented as relative luciferase activity (RLA). (C) HEK293 cells were transiently transfected with ARE-luc and expression vector for AR and treated for 24 h with 10 nM DHT or varying molar concentrations of bavachin as indicated. An untreated group served as a control. These experiments were repeated at least three times.* and ** indicate significant differences from the control at the p<0.05 and 0.001 levels, respectively.

Fig. 4.. Effects of bavachin on endogenous estrogen-responsive PR and pS2 mRNA levels were examined. Cells were exposed to E2 and bavachin for 24 h. The qRT-PCR results for PR, pS2 in MCF-7 cells are shown (n=3).

Fig. 5.. Effects of bavachin on ERα protein levels. (A) MCF-7 cells were treated for 24 h with 10 nM E2 or increasing concentrations of bavachin as shown. An untreated group served as a control. After the incubation, the cells were lysed and total protein extracts were resolved by SDS-PAGE and immunoblotted using an anti-ER antibody or an anti-β-actin antibody. ER densitometry values are expressed as a percentage of the control (down). These experiments were repeated at least three times. * and ** indicate statistically significant differences from the control. (B) MCF-7 cells were treated for 12 h with or without 10 μM bavachin and 10 μM MG132. After the incubation, the cells were lysed and total protein extracts were resolved by SDS-PAGE and immunoblotted using an anti-ERα antibody or an anti-β-actin antibody. ER densitometry valuesare expressed as a percentage of the control (down). These experiments were repeated at least three times. *Indicate statistically significant differences from control.

Fig. 6.. Effects of bavachin on the proliferation of MCF-7 BOS cells. MCF-7 BOS cells were plated in 96 well plates and grown in phenol red-free DMEM containing 5% CD-FBS for 2 days. Cells were treated as indicated and cultured for 6 days. The cell growth was determined by MTT method and expressed as the relative proliferative effect to E2. Data show mean ± SD (n=4).