Emodin Inhibits Breast Cancer Cell Proliferation through the ERα-MAPK/Akt-Cyclin D1/Bcl-2 Signaling Pathway

Background: The aim of the present study was to investigate the involvement of emodin on the growth of human breast cancer MCF-7 and MDA-MB-231 cells and the estrogen (E2) signal pathway in vitro . Materials and Methods: MTT assays were used to detect the effects of emodin on E2 induced proliferation of MCF-7 and MDA-MB-231 cells. Flow cytometry (FCM) was applied to determine the effect of emodin on E2-induced apoptosis of MCF-7 cells. Western blotting allowed detection of the effects of emodin on the expression of estrogen receptor α, cyclin D1 and B-cell lymphoma-2 (Bcl-2), mitogen-activated protein kinases (MAPK) and phosphatidylinostiol 3-kinases (PI3K). Luciferase assays were emplyed to assess transcriptional activity of ERα. Results: Emodin could inhibit E2-induced MCF-7 cell proliferation and anti-apoptosis effects, and arrest the cell cycle in G0/G1 phase, further blocking the effect of E2 on expression and transcriptional activity of ERα. Moreover, Emodin influenced the ER α genomic pathway via downregulation of cyclin D1 and Bcl-2 protein expression, and influenced the non-genomic pathway via decreased PI3K/Akt protein expression. Conclusions: These findings indicate that emodin exerts inhibitory effects on MCF-7 cell proliferation via inhibiting both non-genomic and genomic pathways.


Introduction
Breast cancer is one of the most common malignant tumor.the mortality rate of breast cancer has increased yearly in China since 1991.In addition, the trend will continue to increase (Shi et al., 2014).Breast cancer is a kind of estrogen-dependent malignant tumors.Evidence indicates that estrogen is one of most important pathogenic factors to the formation and development of breast cancer, and its biological activity is dependent on the estrogen receptor-α (ERα) (Kong et al., 2003).ESR-α polymorphisms are associated with susceptibility to breast cancer (Lu et al., 2013).ERα plays an important role in breast cancer initiation and progression and represents a major target in cancer therapy.Izadi's study show that ERα methylation is correlated with poor prognosis subtypes of breast tumors in Iranian patients and may play an important role in pathogenesis of the more aggressive breast tumors (Izadi et al., 2012).The expression and activity of ERα is regulated by multiple mechanisms at the transcriptional and post-translational levels (Coombes et al., 2007).Estrogen interaction of ERα regulates the transcription gene through activating estrogen reactive element (ERE), and regulates the proliferation of breast cancer through MAPK/Erk1/2 and PI3K/Akt signaling RESEARCH ARTICLE Emodin Inhibits Breast Cancer Cell Proliferation through the ERα-MAPK/Akt-Cyclin D1/Bcl-2 Signaling Pathway Jia-Qi Sui, Kun-Peng Xie, Wei Zou, Ming-Jie Xie* pathways (Kaufmann et al., 2007).Selective estrogen receptor modulators (SERMs), such as tamoxifence and raloxifene, can bind to estrogen receptor and exert estrogen-like actions and are widely used in treatment and prevention of postmenopausal osteoporosis, breast cancer and other gynecological diseases.However, SERMs may also be responsible for some potentially very serious adverse effects, such as thromboembolic disorders and immunity suppression.Therefore, finding an effective, safe and less side effect antitumor drug is necessary to overcome this current and urgent gap in therapy.Emodin is isolated and extracted from traditional Chinese drugs rhubarb, polygonum cuspidate and aloe that exhibits high antitumor activity.However, there is no report about emodin in treatment of breast cancer and its involvement in ERα signaling pathway in vitro.The objectives of present study are to investigate if emodin has anti-breast cancer effect through ERα signaling pathway, which will provide a theoretical foundation for development of emodin as antitumor drugs.Cell culture and treatment MCF-7 cells and MDA-MB-231 cells were cultured in RPMI-1640 supplemented with 10% fetal calf serum, penicillin (100U/mL) and streptomycin (100U/mL) at 37℃ in a 5% CO 2 incubator, cells in log phase were used.For E2 treatment, cells were maintained in phenol red-free media with 5% charcoal-stripped FCS for 48h and then in serum-free medium for 24h.After that, cells were treated with different emodin (the final concentrations were 20, 40, 60μmol/L) or/and E2 for 48h before cell harvest.The cells lysates were prepared for Western blot.To test the effects of different inhibitors, all inhibitors and emodin (40μmol/L) were added 24h before the E2 addition.The cells lysates were prepared for Western blot.

MTT cell viability assay
MCF-7 cells at log phase were seeded in 96-well plates at 5×10 3 cells/well.When cells attached to the plate, MCF-7 cells were treated either with different concentrations of emodin (the final concentrations were 10, 20, 40, 60μmol/L) for 48h, or with above concentrations of emodin and E2 for 24/48h.In control experiment, the cells treated with 1% DMSO.Before incubated with E2 and emodin, cells were pretreated with 5% charcoal stripped FCS for 48h, then in instead of phenol red-free serum-free medium for 24h.Then, MTT (20μl at 5 mg/ml) was added to each well and culture was continued for 4h.Medium was then replaced with 150μl of DMSO.Absorption was measured at 490nm with reference wavelength at 570nm.Similar experiment was repeated three times.

Cell cycle and apoptosis analysis
MCF-7 cells were incubated with E2 and different concentrations of emodin for 24h.The Cell suspension was washed with PBS.Then, cells were fixed with 70% ethanol over 24h at 4℃.Finally, cells were treated with 20μg/ml RNase A and 50 μg/ml of propidium iodide for 30 min.Stained cells were monitored by flow cytometry.

Transient transfection experiments
MCF-7 cells were plated in 24-well plates at 1×10 5 cells/well with RPMI-1640 (phenol red free) supplemented with 5% charcoal stripped FCS.The cells were transfected with phenol red-free RPMI-1640 at 95% confluence.The transient transfection was performed using Lipofectam ine TM 2000 according to the manufacturer's directions.Cells were treated in triplicate, 24h later with emodin and E2.The cells were harvested 24h later, and the luciferase activities were assayed.

Statistical analysis
Data were expressed as mean±SD.SPSS13.0 and Excel XP with t-text were used for statistic analysis.A p value of <0.05 was considered significant.

Effects of emodin on E2 induced MCF-7 cell and MDA-MB-231 cell proliferation
We evaluated the effect of emodin on the proliferation of breast cancer cells in vitro, determined by MTT assay.As shown in Figure 1A, emodin inhibits the proliferation of ERα-positive MCF-7 cell and ERα-negative MDA-MB-231 cell in dose-dependent manner.At 48h, the inhibition of 40μmol/L emodin on MCF-7 cell and MDA-MB-231 cell were 38.15±2.93%,24.15±2.43%,respectively (p<0.05).The effect of emodin on MCF-7 cell is more significant.
To understand molecular mechanisms underlying the growth inhibition induced by emodin, we examined the proliferation of E2-induced MCF-7 cell in vitro using MTT assay.It turned out that 10nM E2 significantly increases the proliferation of MCF-7 cell (p<0.and MDA-MB-231 cell with emodin at 40μmol/L for 48h and with E2, cell viability is reduced by 58.90±2.72%,21.58±3.07%(p<0.05, Figure 1B), respectively.This result suggests emodin could inhibit E2-induced cells proliferation effect which is interaction with E2.Based on above results, we selected MCF-7 cells for the subsequent experiments.

The effect of emodin on E2-induced MCF-7 cells cycle progression and apoptosis
As shown in Figure 2, Flow cytometric analysis showed that E2 slightly decreases the number of cells in G0/G1-phase while slightly increases the number of cells in S-and G2/M-phase, there is no significant difference.Combined treatment with 40μmol/L of emodin and 10 nmol/L E2 significantly increases the number of cells in the G0/G1-phase by 28.4±1.82%(p<0.05).In addition, the cell apoptosis rates in control group and E2 group are 13.24±0.67%and 7.93±0.53%,respectively, there is statistical significance (p<0.01, Figure 2B), suggesting that E2 inhibits the apoptosis of MCF-7 cells.This result indicated that emodin inhibits E2-induced cell against apoptosis.Furthermore, combined treatment with emodin and E2 significantly increases the apoptosis rate of MCF-7 cells (Figure 2B, p<0.05).

The effect of emodin on E2-induced ERα target genes Cyclin D1 and Bcl-2 expression
To investigate the signaling pathway for the involvement of ERα in the inhibitory effect of emodin on E2-induced cell growth, we tested the effect of emodin on E2-induced ERα target genes Cyclin D1 and Bcl-2 expression.In E2-treated cells, the expression of ERα target genes Cyclin D1 and Bcl-2 are obviously increased compared with control (p<0.01; Figure 4).However, the increases of Cyclin D1 and Bcl-2 are significantly inhibited by 20 µmol/L emodin (p<0.01) and further reversed by 40 µmol/L emodin (87.34±0.83%,85.48±0.86%,p<0.01).

The effect of emodin on AKT and MAPK activation induced by E2
To investigate the possible involvement of emodin in E2- dependent rapid activated pathways, we studied the AKT and MAPK activation by examine their phosphorylation status.E2 increases the phosphorylation of AKT and MAPK (p<0.01, Figure 5).The phosphorylation of AKT and MAPK is significantly reduced by specific inhibitor PD-98095 and LY-294-002, respectively (p<0.01; Figure 5).Furthermore, the expression of p-AKT and p-MAPK is reduced of 29.89±2.49%(p<0.05),63.42±3.04%(p<0.01) by 40 µmol/L emodin.

Discussion
As the nature anthraquinone compounds found in rhubarb, polygonum, cuspidate and aloe, emodin was found to have effect of anti-cancer for different stages of cancer development (Liu et al., 2006), He et al. (2012) have showed that emodin extracted from Chinese herbs may inhibited cell proliferation in NSCLC cells by downregulation ERCC1 and Rad51 (He et al., 2012).All of this indicating that emodin could be potentially developed for clinical applications.Recently, emodin has been a focal point in the development of breast cancer therapy.Our study indicates that emodin inhibits the proliferation of ER-positive MCF-7 cell and ER-negative MDA-MB-231 cell in a dose-dependent manner, in which the inhibition of MCF-7 cell is more significant.It has been indicated that ERα regulates genomic and non-genomic mechanism in breast cancer initiation and progression.Mechanistically, dysregulation of ERα signaling pathway contributes to the onset and progression of breast cancer (Yamaguchi, 2007;Ma et al., 2012).Here we show that emodin inhibits the effects of ERα on MCF-7cells.Emodin may exert its effect through associating with ERα.Our results indicate emodin could inhibit E2-activated ERα transactivation activation, suggesting that ERα is a key target for drugs.
The genomic mechanism of ER signaling is direct action of E2 bound to ERα.This is followed by ERα dimerization, binding to estrogen-responsive genes (ERE).Increased transcription of these genes enhances mammary epithelial cell proliferation and tumorigenesis, thereby regulates the transcription of target gene (Yao et al., 2010).In breast cancer tissues, several estrogen response proteins containing ERE directly promote breast cancer cells proliferation and survival, we main talk about Cyclin D1 and Bcl-2.CyclinD1 is a kind of cell cycle regulator protein which is closely related to tumorigenesis (Takahashi-Yanaga et al., 2008).Bcl-2 is the most important regulator protein of apoptosis protein family, playing the main role in tumor cells to prevent apoptosis and promote cell survival, Expression of Bcl-2 in tumor cells was strongly associated with expression of estrogen and progesterone receptors (Borner et al., 2004;Jaafar et al., 2012).Our results indicate that emodin significantly inhibit the E2-induced expression of ERα target genes Cyclin D1 and Bcl-2, and arrested MCF-7 cells at G0/G1 phase, suggesting that emodin may inhibit tumor play antitumor effect through the down-regulation of Cyclin D1 and Bcl-2.
The non-genomic mechanism of E2 plays an important role in the progression and apoptosis of tumor cells.E2 activates transcriptional activity via a number of pathways including PI3K/Akt, MAPK/Erk1/2 pathway which exists in cell membrane.Furthermore, it has been suggested that E2 could rapidly bind to receptors related to the development and progression of tumor and result in its phosphorylation, the effect is very rapid and not related to gene expression (Chakraborty et al., 2010;Mendoza et al., 2011).Our data are supported by previous studies showing that emodin exerts its anticancer effects via inhibiting PI3K/Akt and MAPK/Erk1/2 pathways.Therefore, ERα is directly involved in the inhibition of PI3K/AKT by emodin through regulating non-genomic pathway, this hypothesis is consistent with Zhang's study (2005), which showing that E2 mediates rapid non-genomic pathway through stimulating IGF-IR in breast cancer cells, thereby activating PI3K/Akt signaling pathway, promoting breast cancer cells proliferation and differentiation to exert its biological effect.