Figure 2. Phospho-proteomic profiling of E2-BSA and puerarin effects on ERK1/2 phosphorylation in ESCs and confirmed by western blot analysis. 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 (1028 mol/L) for 15 min. AKT, Chk2 and ERK1/2 were activated by E2-BSA. Expression was normalized to total ERK1/2. doi:10.1371/journal.pone.0045529.g002 severe side effects; therefore, medical treatment with this agent is a good option for avoiding disease relapse after the initial surgical and/or medical therapy. To further elucidate the general mechanism of puerarin action against estrogen, the experiments were carried out on ESCs which were from kinds of patients with no specificity. First, we focused on the ability of puerarin to bind to ERs on viable cells, so the whole cell ER competitive binding assay was performed. This assay has the advantage of providing the means to rapidly and reproducibly quantitate puerarin and E2 specific binding in small samples of whole cells in culture [21] and it also imitates the local circumstance in vivo. The results showed that puerarin has the ability to compete with estrogen at the ERs on whole ESCs. In estrogen-dependent disease, the classical pathway by which estrogen induces cell proliferation is through binding to a ligand- dependent ER, thus inducing its conformational change and subsequently interacting with EREs, together with multiprotein complexes of transcription factors and co-activators, to activate transcription [22]. In addition to this classic genomic effect of estrogen, several non-genomic actions have also been reported that initiate at the cell surface through binding of estrogen to membrane receptors [23], including calcium mobilization [24–25], cAMP stimulation [26], phospholipase C activation and inositol phosphate genera- tion [27], and MAPK activation [28]. Since puerarin has the ability to compete with estrogen for the ERs, it was of interest to us to ascertain whether puerarin could antagonize the effect of estrogen to cause proliferation through rapid non-genomic pathways. To distinguish non-transcriptional signaling pathways, E2-BSA, a macromolecular complex considered to be membrane-imperme- able, was used, since this is prevented from reaching the classical ERs in the nucleus [29–31]. Human phospho-kinase array experiments showed that E2-BSA induced significant phosphory- lation of several kinases, including AKT, Chk2 and ERK1/2, whereas puerarin weakened its effect. By western blotting, we confirmed that E2-BSA-induced activation of ERK1/2 in ESCs was transient, reaching a peak at 15 min before returning to basal levels within an hour. This observation allows us to propose that Puerarin Suppresses Proliferation of ESCs Figure 3. Western blot analysis of E2-BSA and puerarin effects on ERK1/2 phosphorylation in ESCs. Cells were treated as indicated. A. Concentration-dependent effects of E2-BSA on ERK1/2 phosphorylation in ESCs. Cells were treated with vehicle (DMSO) or E - 2 BSA (10210 to 1026 mol/L) for 15 min. B. Time-dependent effects of E2- BSA on ERK1/2 phosphorylation in ESCs. Cells were treated with 1028 mol/L of E2-BSA for 0, 15, 30, 45 or 60 min. C. ESCs were treated with 1028 mol/L of E2-BSA, 1029 mol/L puerarin or both, as indicated; in some groups, cells were pretreated with 20 mmol/L of U0126 or 1 mmol/ L of ICI182,780 for 30 min prior to the stimulation. Expression was normalized to total ERK1/2. doi:10.1371/journal.pone.0045529.g003 this stimulating phenomenon initiates at the plasma membrane, and does not involve ER-mediated gene transcription. ERK, one of three well-characterized subfamilies of MAPK, phosphorylates specific tyrosine and threonine residues of target protein substrates, and regulates cellular activities that include gene expression, mitosis, movement, metabolism, survival, and programmed cell death [32–33]. Our data that puerarin reduced ERK1/2 phosphorylation in ESCs treated with E2-BSA support our proposition that puerarin could exhibit anti-estrogenic activity partly through inhibiting of the ERKs signaling pathway. Further evidence that membrane receptors are involved in the non- genomic effects of E2-BSA is provided by the observation that when ESCs were treated with the ER antagonist, ICI182,780, phosphorylation of ERK1/2 was inhibited. Meantime, puerarin can’t reverse the phosphorylation of ERK1/2 on this occasion. These phenomenons indicated that puerarin inhibited activation of ERK through competing with estrogen to bind to membrane receptors initiated MAPK signaling (Fig. 3C). We found that all the doses of E2-BSA we tested could promote proliferation of ESCs as shown in figure 4A and various concentraions (from 10210 mol/L to 1024 mol/L) of puerarin could reverse this effect by CCK-8 assay and that 1029 mol/L achieved the most useful effect (data not shown). When U0126, a ‘‘small molecule’’ inhibitor of MEK1/2, the upstream activating kinases of ERK1/2, was added, ESCs proliferation couldn’t be promoted by E2-BSA anymore, and even the proliferation was much slower than the DMSO treatment group (Fig. 4C). These results demonstrated that E2-BSA promoted ESCs proliferation PLOS ONE | www.plosone.org 4 September 2012 | Volume 7 | Issue 9 | e45529