Estrogen receptor modulators genistein, daidzein and ERB-041 inhibit cell migration, invasion, proliferation and sphere formation via modulation of FAK and PI3K/AKT signaling in ovarian cancer

Soy isoflavones 5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one (genistein, G6776), 7-hydroxy-3-(4-hydroxy-phenyl)-4H-1-benzo-pyran-4-one, (daidzein, D7802) and the selective estrogen receptor modulator (SERM) 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3-benzoxazol-5-ol (ERB-041, PZ0183), were purchased from Sigma-Aldrich (St Louis, MO) and dissolved in dimethyl sulfoxide (DMSO).

Ovarian cancer cell lines SKOV-3, A2780CP and OVCAR-3 were used in this study. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Invitrogen) and antibiotics (50 U/ml penicillin and 50 µg/ml streptomycin) (Invitrogen) in a humidified atmosphere containing 95% air and 5% CO₂ at 37 °C [15, 20]. SKOV-3 and OVCAR-3 were purchased from American Type Culture Collection (ATCC; Manassas, VA). A2780CP was kindly provided by Prof. B. Tsang, Department of Obstetrics and Gynecology, University of Ottawa).

Cells plated in 6-well plates were treated with 10 (low pharmacological) and 50 µM (high pharmacological) genistein and daidzein and 0.01, 0.1 and 10 µM ERB-041 in complete medium. Control cells were treated with an equal amount of DMSO. After 24 h of treatment, cells were washed with PBS and harvested with Mammalian Cell Lysis Reagent CelLytic™ M (Sigma, C2978) containing protease inhibitor cocktail (Sigma, P8340), phosphatase inhibitor cocktail 2 (Sigma, P5726) and phosphatase inhibitor cocktail 3 (Sigma, P0044). After 15 min of incubation on a shaker at 4 °C, cells were scraped and centrifuged. The protein-containing supernatant was collected. 30 µg protein lysate was separated by 7.5% SDS-PAGE and electroblotted to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% skim milk and then hybridized with primary antibodies: anti-ERα (Dako, CA, M3634), anti-ERβ (Santa Cruz, sc-8974), anti-p-FAK (Cell Signaling, Danvers, 8556), anti-FAK (Santa Cruz, sc-558), anti-p-PI3K85 (Abcam, ab138364), anti-p-AKT (Cell Signaling, 4060), anti-AKT (Cell Signaling, 4691), anti-p-GSK3β (Cell Signaling, 5558), anti-GSK3β (Cell Signaling, 12456), anti-p21 (Santa Cruz, sc-397), anti-cyclin D1 (Santa Cruz, sc-753), anti-E-cadherin (Cell Signaling, 3195), anti-Vimentin (Cell Signaling, 5741) and anti-Actin (Sigma, A5060) at 4 °C overnight. The membranes were then washed and probed with appropriate secondary antibodies conjugated with horseradish peroxidase (Santa Cruz) at room temperature for 1 h. After washing, the blots were detected with Clarity Western Enhanced Chemiluminescence Substrate (Bio-rad) and visualized by autoradiography [15, 20].

For genistein, daidzein and ERB-041 treatments, cells were treated with 10 and 50 µM genistein and daidzein, 0.01, 0.1 and 10 µM ERB-041 or DMSO. For transient knockdown of ERα and ERβ in SKOV-3, cells were transfected with siRNAs of ERα (Santa Cruz, Santa Cruz, CA, sc-29305) and ERβ (Santa Cruz, sc-35325) using SilentFect™ (Bio-Rad Laboratories, Hercules, CA) for 48 h before cell counting and cell plating. Control siRNA (sc-37007) was used as control. Cells (1.25 × 10⁵) were plated in DMEM medium on the upper compartment of a Transwell chamber. Then, cells were migrated through an 8-µm pore size membrane or invaded through a Matrigel-coated membrane (Corning^® BioCoat™ Matrigel^® Invasion Chambers) toward lower chamber with DMEM plus 10% FBS (as a chemoattractant). After 8–24 h, cells on the upper side of the membrane were removed with a cotton bud and the migrated or invaded cells were then fixed, stained, and counted under a light microscope [20, 21].

Cell proliferation was determined by XTT assay and foci formation assay [15]. For XTT assay, 3000 cells per wells were seeded in 96-well plates overnight. Cells were washed with PBS and treated with 1, 5, 10 and 50 µM genistein and daidzein, 0.01, 0.1 and 10 µM ERB-041 or DMSO in complete medium. Cell proliferation was measured 24 and 48 h after treatment using Cell Proliferation Kit II (Roche Biosciences, Indianapolis, IN, USA) according to manufacturer’s instructions in an Infinite^® 200 microplate reader at 492 nm (Tecan Group Ltd, Männedorf, Switzerland).

For foci formation assay, A2780CP cells per wells were seeded in 6-well plates at a density of 500 cells per well overnight. Cells were washed with PBS and treated with 10 and 50 µM genistein and daidzein, 0.01, 0.1 and 10 µM ERB-041 or DMSO in complete medium. At day 7 after treatment, cells were fixed and stained with 1% crystal violet (Sigma-Alrich). Numbers of foci were counted.

SKOV-3 cells were treated with 50 µM genistein and daidzein, 10 µM ERB-041 or DMSO for 48 h. Harvested cells were washed in PBS and fixed in cold 70% ethanol at 4 °C overnight. Cells were then washed, treated with 50 μl RNase (100 μg/ml, Sigma) and stained with 200 μl propidium iodide (50 μg/ml, Invitrogen) in dark for 20 min. Green fluorescent-stained cells were analyzed by flow cytometry using a FACSCanto flow cytometer (BD, San Jose, CA) and FlowJo software. Cell-cycle distribution was presented as percentage of the G1, S, and G2/M phases of cells.

SKOV-3 cells were treated with 50 µM genistein and daidzein, 10 µM ERB-041 or DMSO for 48 h. Adherent and floating cells were harvested, washed in PBS and stained with Annexin-V-FLUOS Staining Kit (Roche) according to manufacturer’s protocol. The double-stained cells were then analyzed by flow cytometry using a FACSCanto flow cytometer (BD).

OVCAR-3 sphere forming cells established by suspension culture in stem cell-condition medium (serum-free DMEM/F12 supplemented with 20 ng/ml human recombinant epidermal growth factor, 10 ng/ml human recombinant basic fibroblast growth factor and antibiotics penicillin and streptomycin) (Invitrogen) using ultra-low attachment 6-well plates (Corning) were treated with genistein (10 µM), daidzein (10 µM), ERB-041 (0.1 and 10 µM) or DMSO for 7 days. Spheres generated were photographed and sphere number were counted under light microscope [22].

We first examined the expression of ERα and ERβ in three ovarian cancer cell lines, SKOV-3, OVCAR-3 and A2780CP by Western blot analysis (Fig. 1). ERα was expressed in SKOV-3, but not in OVCAR-3 and A2780CP, whereas ERβ was expressed in all three cell lines.

Next, we determined the effect of genistein, daidzein and ERB-041 on ovarian cancer cell migration and invasion. Transwell migration and invasion assays revealed significantly reduced migration and invasion in genistein and daidzein treated cells compared with that in control cells dose-dependently in both SKOV-3 and A2780CP cells (all p < 0.05) (Fig. 2). Low (10 µM) and high (50 µM) doses of genistein inhibited cell migration by 65 and 77% in SKOV-3 cells and 54 and 68% in A2780CP cells, respectively (Fig. 2a, left panel), whereas 10 and 50 µM of daidzein inhibited cell migration by 63 and 75% in SKOV-3 cells and 49 and 64% in A2780CP cells, respectively (Fig. 2b, left panel). The inhibition on cell invasion was 76 and 82% in SKOV-3 cells and 41 and 66% in A2780CP cells by 10 and 50 µM genistein, respectively (Fig. 2a, right panel), whereas the inhibition was 62 and 78% in SKOV-3 cells and 43 and 66% in A2780CP cells by 10 and 50 µM daidzein, respectively (Fig. 2b, right panel).

Significantly reduced cell migration and invasion in ERB-041 treated cells was also demonstrated by Transwell migration and invasion assays when compared with that in control cells in SKOV-3, A2780CP and OVCAR-3 cells (all p < 0.05) (Fig. 3). ERB-041 significantly reduced cell migration by 68% at the minimum dose of 0.01 µM in A2780CP cells, 43 and 42% at 0.1 µM in SKOV-3 and OVCAR-3 cells, respectively (Fig. 3a). 40, 54 and 37% inhibition on cell migration was detected in SKOV-3, A2780CP and OVCAR-3 cells by 10 µM ERB-041 treatment, respectively (Fig. 3a). At the minimum dose of 0.01 µM ERB-041 in A2780CP and OVCAR-3 cells, 57 and 29% reduction on cell invasion was demonstrated, respectively (Fig. 3b). 10 µM ERB-041 significantly reduced cell invasion by 50, 60 and 42% in SKOV-3, A2780CP and OVCAR-3 cells, respectively (Fig. 3b).

We showed the inhibitory effect of genistein, daidzein and ERB-041 on ovarian cancer cell migration and invasion. We next examined if knockdown of ERβ would display opposite effect. mRNA and protein expression of ERβ was evaluated by qPCR and immunoblotting at 48 and 72 h after siRNA transfection, respectively. siERβ profoundly reduced ERβ mRNA and protein expression in SKOV-3 cells relative to the non-target control siRNA cells (Fig. 4a). Silencing of ERβ significantly increased cell migration and cell invasion (Fig. 4b). We also performed transient knockdown of ERα in SKOV-3 cells and found that silencing of ERα had no significant change on cell migration and invasion (Additional file 1: Figure S1).

To determine if the inhibitory migration and invasion effect of genistein, daidzein and ERB-041 is due to reduced cell proliferation, XTT assay was performed 24 and 48 h after genistein, daidzein and ERB-041 treatment in SKOV-3, A2780CP or OVCAR-3 cells. We found that genistein, daidzein and ERB-041 inhibited cell proliferation in a dose- and time-dependent manner (Fig. 5a–c). Although we found 50 µM of genistein and daidzein and 0.1 and 10 µM ERB-041 showed significantly inhibitory effect on cell proliferation ranging from 11 to 26% in SKOV-3, A2780CP or OVCAR-3 cells (all p < 0.05), their effects on reduction of cell migration and invasion are much more prominent (Figs. 2, 3, 5).

Besides XTT assays, we applied focus formation assay to investigate the effect of genistein, daidzein and ERB-041 on ovarian cancer cell proliferation. We found the number of colonies from genistein and daidzein treated A2780CP cells decreased dose-dependently (Fig. 5d). 10 µM genistein and daidzein could cause significant inhibition by 20 and 16%, respectively (Fig. 5d). Genistein and daidzein at 50 µM caused 54 and 38% decrease on colonies formation, respectively (Fig. 5d). 0.1 µM ERB-041 did not have significant effect on focus formation, whereas 10 µM inhibited focus formation by 20% (Fig. 5d).

We next investigated the effect of genistein, daidzein and ERB-041 on cell cycle progression in SKOV-3 cells by flow cytometry analysis using PI staining. 50 µM genistein induced S and G2/M cell cycle arrest, whereas 50 µM daidzein and 10 µM ERB-041 induced G1 cell cycle arrest (Fig. 6).

The apoptosis effect of genistein, daidzein and ERB-041 in SKOV-3 cells was analyzed by flow cytometry analysis using Annexin V/PI double staining. The percentage of apoptotic cells was significantly increased in 50 µM genistein (17%), 50 µM daidzein (9.6%) and 10 µM ERB-041 (10.9%) when compared to control cells (4.2%) (Fig. 7).

Our finding that genistein, daidzein and ERB-041 could reduce ovarian cancer migration and invasion prompted us to examine their roles on the regulation of stem-like properties because metastasis was one of the characteristics of CSCs. We then evaluated the capacity of OVCAR-3 cells treated with genistein (10 µM), daidzein (10 µM), ERB-041 (0.1 and 10 µM) or DMSO (vehicle) to grow as multi-cellular spheroids in stem-condition culture systems by sphere formation assay. Results showed that genistein and daidzein treated OVCAR3 cells formed smaller and less sphere cells (50 and 32% decrease, respectively) than that of control cells (all p < 0.05) (Fig. 8a). Dose dependent inhibitory effect was observed in ERB-041 treated cells (Fig. 8b). Smaller and less sphere cells (24 and 58% decrease, respectively) were formed in 0.1 and 10 µM treated OVCAR-3 cells compared with that of control cells (all p < 0.05) (Fig. 8b). The inhibition of ovarian cancer sphere formation suggested that genistein, daidzein and ERB-041 could regulate stem-like properties in ovarian cancer cells.

Next, we investigated the possible downstream signaling by which genistein, daidzein and ERB-041 mediated their effect on ovarian cancer by immunoblotting. Focal adhesion kinase (FAK) is a non-receptor protein tyrosine kinase that has a critical role on cancer cell migration and invasion [23]. AKT is a serine/threonine kinase that plays an important role in many cellular processes, such as cancer cell proliferation, survival, migration and stem cell regulation [24]. We found that genistein, daidzein and ERB-041 treated SKOV-3 cells reduced FAK and AKT activation as detected by phosphorylation on Tyr³⁹⁷ and Ser⁴⁷³, respectively (Fig. 9). Moreover, genistein, daidzein and ERB-041 also suppressed phosphorylation of Phosphatidylinositol 3 kinase (PI3K)85 and GSK3β, upstream and downstream of AKT respectively [25, 26], suggesting that genistein, daidzein and ERB-041 could modulate PI3K85/AKT/GSK3β signaling in ovarian cancer (Fig. 9). We also examined expression of cell cycle regulators p21 and cyclin D1 [27, 28] as well as epithelial–mesenchymal transition (EMT) markers E-cadherin and Vimentin [29] in genistein, daidzein and ERB-041 treated cells. We detected increased p21 and E-cadherin, and reduced vimentin expression in genistein treated SKOV-3 cells (Fig. 9). Daidzein and ERB-041 treated cells decreased cyclin D1 expression, but have no virtual effect on E-cadherin and Vimentin expression (Fig. 9).