Morin hydrate, bicinchoninic acid (BCA) solution, TPA, hematoxylin, eosin, and sulforhodamine B (SRB) were purchased from Sigma-Aldrich; Merck KGaA. DMEM, insulin solution, trypsin and antibiotic/antimycotic solution were acquired from Welgene, Inc. Trichloroacetic acid (TCA) was purchased from Samchun Pure Chemical Co., Ltd., and 30% polyacrylamide solution, protease inhibitor cocktail, and phosphatase inhibitor cocktail were obtained from GenDEPOT. Primary antibodies for Akt (cat. no. 4691), phospho-Akt (cat. no. 4060), ERK1/2 (cat. no. 4695), phospho-ERK1/2 (cat. no. 4370), JNK (cat. no. 9258), phospho-JNK (cat. no. 4668), p38 mitogen-activated protein kinase (MAPK; cat. no. 8690), phospho-p38 MAPK (cat. no. 4511), NF-κB (cat. no. 8242), phospho-NF-κB (cat. no. 3033), c-Jun (cat. no. 9165), c-Fos (cat. no. 2250), glycogen synthase kinase (GSK)-3β (cat. no. 5676), phospho-GSK-3α/β (cat. no. 9331) and GAPDH (cat. no. 5174) were purchased from Cell Signaling Technology, Inc., and goat anti-rabbit immunoglobulin G-horseradish peroxidase-conjugated antibody (cat. no. 31460) was from Thermo Fisher Scientific, Inc. PI3K/Akt inhibitors LY294002 and wortmannin were obtained from LC Laboratories and Cayman Chemical Company, respectively. Primers for reverse transcription-quantitative PCR (RT-qPCR) were synthesized by Bioneer Corporation.

MCF-7 human breast cancer cells were obtained from the Korean Cell Line Bank. The cells were grown in DMEM supplemented with 10% FBS (American Type Culture Collection), 10 µg/ml insulin and 1% antimycotic/antibiotic mixture at 37°C in 5% CO₂ atmosphere. The cells were serum-starved for 24 h prior to the addition of conditioned DMEM containing morin hydrate (0-200 µM) and/or 100 nM TPA at 37°C in 5% CO₂ atmosphere. All cell culture experiments were performed at 37°C in a 5% CO₂ atmosphere in DMEM.

A total of 5,000 cells were seeded into a 96-well plate in each well and attached for 24 h in DMEM supplemented with 10% FBS. Then, DMEM supplemented with 10% FBS in each well was replaced with conditioned DMEM containing 5% FBS and various concentration of morin hydrate. The cells were further incubated for 24, 48 or 72 h; then the conditioned DMEM was removed. The cells were fixed with 20% TCA solution for 1 h, washed with tap water, and dried for 2 h, all at room temperature. To stain the dried cells, 0.4% SRB solution was added into each well and stored at room temperature for 30 min. The stained cells were rinsed with 1% acetic acid and dried at room temperature. Then, 100 µl of 10 mM Tris-HCl (pH 5.0) buffer was added in each well to dissolve SRB, and absorption was measured at a wavelength of 510 nm by a SpectraMax^® M2e microplate reader (Molecular Devices, LLC).

A total of 5×10⁵ cells/well were seeded into collagen-coated six-well plates and attached for 24 h in DMEM supplemented with 10% FBS. Then, the cell monolayers were scratched by 1 ml micropipette tip and DMEM supplemented with 10% FBS was removed by aspiration. Then, the cell monolayers were washed by PBS to remove detached cells and serum-starved for 24 h in serum-free DMEM. Then, serum-free DMEM was removed and the cell monolyers were treated with various concentration of morin hydrate dissolved in DMEM containing 1% FBS for 1 h prior to taking photographs. Then, the cells were further administrated with TPA to trigger cell migration for 24 h and the scratch zones were observed and photographed. The change of the width of the scratch zone was observed and images were captured with Nikon light microscope (Nikon Corporation).

A total of 3×10⁴ cells were plated in the upper chambers of 24-well transwell plates (8-µm-pore; Corning Inc.) in cold serum-free DMEM. For the invasion assay, plates were coated with the Matrigel^® (BD Biosciences) for 1 h. DMEM supplemented 10% FBS were added in the lower chambers; then, the cells were cultured for 24 h in the upper chamber. The media in the upper chambers were replaced with serum-free DMEM and various concentrations of morin hydrate. The media in lower chambers were replaced with new DMEM supplemented with 10% FBS. After 1 h, TPA was added into the upper chambers as an inducer for invasion. The cells were further incubated for 24 h at 37°C in 5% CO₂ atmosphere. The cells that migrated through or invaded the polycarbonate filter were fixed with 100% methanol and stained with heamatoxylin and eosin Y solution for 10 min. The stained cells were observed with light microscope and images were captured using an inverted optical microscope. ImageJ (version 1.6.0_20) was used for data analysis (National Institutes of Health).

A total of 5×10⁵ cells/well were attached for 24 h and then further cultured for 24 h in FBS-free DMEM for serum-starvation. The cells were treated with various concentrations of morin hydrate with 100 nM TPA for 24 h. The DMEM were electrophoresed with 8% non-denaturing SDS-PAGE containing 0.1% (v/v) gelatin. The gel was washed with 0.25% Triton X-100 solution to remove SDS and incubated at 37°C in a reaction buffer [5 mM CaCl₂, 0.04% NaN₃ and 50 mM Tris-HCl] overnight to confirm the gelatinase activity of MMP-9. Bands were visualized by Coomassie brilliant blue R staining for 1 h and images were captured with LAS-4000 image analyzer (Fujifilm). ImageJ was used for data analysis.

A total of 5×10⁵ cells/well were attached for 24 h and then further cultured for 24 h in FBS-free DMEM for serum-starvation. The cells were pretreated for 24 h with various concentrations of morin hydrate and further incubated with or without Akt inhibitors, LY294002 (20 µM) and wortmannin (20 µM), for 30 min prior to TPA treatment for 1 h at 37°C. Then, the cells were harvested and analyzed by western blotting.

The cells were treated with morin hydrate and 100 nM TPA for 24 h after 24-h attachment and 24-h serum-starvation. To isolate total RNA, the cells were harvested by trypsinization, collected by centrifugation at room temperature for 3 min at 1,000 × g and lysed with easy-BLUE™ Total RNA Extraction kit (Intron Biotechnology, Inc.). The extraction of total RNA from the lysed cells was performed in accordance with the manufacturer’s protocol. Total RNA (1 µg) was used for cDNA synthesis. RT reaction was performed to synthesize cDNA in 1X GoScript™ reaction buffer containing 2 mM MgCl₂ with GoScript Reverse Transcriptase, 0.5 mM dNTPs (Promega Corporation) and pd(N)9 Random Primers (Takara Bio, Inc.). qPCR was performed using Q Green Sybr Green Master Mix kit (CellSafe Co., Ltd.) using Eco™ Real-Time PCR system (Illumina, Inc.). The following thermocycling conditions were used: Initial denaturation at 95°C for 5 min; 45 cycles of 95°C for 10 sec, 60°C for 20 sec and 72°C for 20 sec. Relative mRNA expression was automatically calculated using EcoStudy software (version 5.0.49; Illumina, Inc.). The primer sequences for PCR amplification are presented in Table I.

A total of 5×10⁵ cells/well were attached for 24 h and then further cultured for 24 h in FBS-free DMEM for serum-starvation. The cells were pretreated with or without morin hydrate for 24 h and then further incubated with or without TPA for 1 h with serum-free DMEM. Then, DMEM was removed, the cells were washed twice with ice-cold PBS and treated with a hypotonic buffer (20 mM Tris-HCl pH 7.4, 10 mM NaCl and 3 mM MgCl₂) containing phosphatase and protease inhibitor cocktails. The cells were collected using a rubber policeman scraper, transferred to new microtubes and incubated for 15 min on ice. Then, 1/8 vol 10% NP-40 (Sigma-Aldrich; Merck KGaA) was added and the cells were vortexed for 10 sec at the highest speed setting. The cells were incubated on ice for 10 min and centrifuged at 2,500 × g for 10 min at 4°C. The supernatants were removed as the cytosolic fraction, and the pellet was lysed for 30 min on ice with the Cell Extraction Buffer (Invitrogen; Thermo Fisher Scientific, Inc.) containing phosphatase and protease inhibitor cocktails. The lysates were separated by centrifugation at 14,000 × g for 30 min at 4°C and the supernatants were removed as the nuclear fraction.

A total of 5×10⁵ cells/well were attached for 24 h and then further cultured for 24 h in FBS-free DMEM for serum-starvation. The cells were pretreated with or without morin hydrate for 24 h and then further incubated with or without TPA for 30 min with serum-free DMEM. Then, DMEM was removed, the cells were washed twice with ice-cold PBS, and whole cell lysates were prepared with radioimmunoprecipitation assay lysis buffer (Biosesang) supplemented with protease inhibitor and phosphatase inhibitor cocktails. The lysed cells were centrifuged at 14,000 × g for 10 min at 4°C, and the supernatants were collected as the whole cell lysate and stored at -80°C until further use. Equal amounts (20 µg) of protein form the whole cell lysate, nuclear fraction or cytosolic fraction were subjected to 8 or 10% SDS-PAGE and transferred to PVDF membranes (Pall Life Sciences). Blocking was performed with 5% non-fat dry milk or 1% bovine serum albumin (BSA; Santa Cruz Biotechnology, Inc.) in Tris-buffered saline-Tween (TBST; 50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) solution for 1 h at room temperature. The primary antibodies for target proteins were diluted at 1:3,000 in 5% non-fat dry milk or 1% BSA in TBST solution, and the reactions for primary antibodies were conducted overnight at 4°C. Subsequently, the membrane was washed by TBST solution, covered with secondary antibody diluted at 1:5,000 in TBS solution and incubated for 1 h at room temperature. The target protein bands were detected by a chemiluminescent substrate [100 mM Tris (pH 8.5; BioShop Canada, Inc.), 1.25 mM luminol, 198 µM coumaric acid and 0.01% hydrogen peroxide (all Sigma-Aldrich; Merck KGaA)] and images were captured using LAS-4000 image analyzer. The band densities were analyzed with Scion Image software (Alpha 4.0.3.2; Scion Corporation).

Data were analyzed using one-way ANOVA followed by Turkey post hoc test, using SPSS software (version 20.0; IBM Corp.). The values are presented as the mean ± SD. P<0.05 was considered to indicate a statistically significant difference.

Previous studies showed that morin hydrate had no cytotoxic activity in KEL FIB human fibroblast cells and EA.hy 926 human umbilical endothelial cells (31,32). Therefore, the current study firstly tested the effects of morin hydrate on cell viability in MCF-7 human breast cancer cells using an SRB colorimetric assay. MTT assay was not used, as the absorbance of insoluble formazan produced by MTT reagents is affected by morin hydrate. The result of the current study showed that cell viability of MCF-7 cells was slightly decreased after treatment with 0-200 µM morin hydrate and the maximum reduction rate of >20% was observed after treatment with 200 µM morin hydrate for 72 h (Fig. 1A). Furthermore, the reduction of cell viability was associated with the delayed cell growth (Fig. 1B). These results suggested that morin hydrate had a weak antiproliferative activity when administered at a concentration range of 150-200 µM in MCF-7 hormone receptor-positive human breast cancer cells.

The current study assessed whether morin hydrate could prevent TPA-induced metastatic potential in MCF-7 human breast cancer cells using cell migration and invasion assays. The result showed that TPA-enhanced cell migration was decreased by morin hydrate treatment in a concentration-dependent manner (Fig. 2A and C). Furthermore, the invasiveness promoted by TPA was also reversed by the presence of morin hydrate in MCF-7 cells in a concentration-dependent manner (Fig. 2B and D). These results suggest that morin hydrate could suppress the TPA-induced metastatic potential in MCF-7 cells.

As indicated above, morin hydrate decreased the invasiveness of MCF-7 cells induced by TPA. Therefore, the current study further evaluated the effects of morin hydrate on MMP-9 activity and expression levels of MMP-9, uPA, uPAR, fibronectin and MMP-7. According to the gelatin zymographic analysis, the proteolytic activity of MMP-9 enhanced by TPA was significantly suppressed by 150 and 200 µM morin hydrate treatment in MCF-7 cells (Fig. 3A). Furthermore, MMP-9, uPA, uPAR and MMP-7 transcription levels were also decreased by morin hydrate supplementation in a concentration-dependent manner (Fig. 3B and C). In addition, gene expression of fibronectin, a known EMT factor, was significantly reduced by morin hydrate treatment (Fig. 3C). However, TPA did not affect the expression of E-cadherin and N-cadherin which are also EMT factors (data not shown). The results suggested that morin hydrate could prevent the participation of MMP-9 activity induced by TPA via negative regulation of MMP-9, uPA, uPAR, fibronectin and MMP-7.

The current study evaluated the effects of morin hydrate on the phosphorylation levels of Akt, MAPK and GSK3β, as well as on the expression levels of AP-1 subunits c-Jun and c-Fos. The result showed that morin hydrate suppressed the phosphorylation of Akt and GSK-3β, in addition to the expression of c-Fos induced by TPA, in a concentration-dependent manner (Fig. 4A-C). By contrast, phosphorylation levels of ERK1/2 and NF-κB, and the expression of c-Jun, which are also involved in the regulation of expression of metastasis-enhancing factors, were not affected by morin hydrate treatment (Fig. 4C and D). JNK and p38 phosphorylation levels were also not altered by treatment with morin hydrate (data not shown). These results suggest that the inhibition of expression of metastasis-enhancing factors by morin hydrate may be associated with the reversal of Akt and GSK3β phosphorylation and inhibition of c-Fos expression.

Therefore, the current study assessed the effect of morin hydrate on the nuclear localization of c-Fos and c-Jun. Morin hydrate decreased the nuclear c-Fos level increased by TPA, in a concentration-dependent manner (Fig. 5A and B). However, the nuclear c-Jun level was not changed by morin hydrate treatment (Fig. 5A). These results imply that morin hydrate could inhibit AP-1 transcriptional activity by decreasing the c-Fos level in the nucleus. The change of the nuclear c-Fos level was also associated with altered whole cell c-Fos expression.

The results of the current study revealed that morin hydrate inhibited Akt and GSK-3β phosphorylation, and c-Fos expression (Fig. 6A), without the change of ERK and NF-κB phosphorylation (Fig. 4D). Therefore, the current study tested the effects of PI3K/Akt inhibitors, LY294002 and wortmannin, on Akt and GSK-3β phosphorylation and c-Fos expressions. The PI3K/Akt inhibitors downregulated the TPA-induced Akt and GSK-3β phosphorylation as well as the c-Fos expression. These results suggested that the inhibitory effect of morin hydrate on the metastatic potential may be regulated by the inhibition of the PI3K/Akt-mediated GSK-3β phosphorylation and c-Fos expression (Fig. 6B).