Morin (Fig. 1A) was obtained from Aging Tissue Bank (Pusan, Korea). Antibodies against N-cadherin, phospho-Akt, Akt, phospho-GSK3β and GSK3β were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-biphenyl tetrazolium bromide (MTT), 4′,6-diamidino-2-phenyindole, dilactate (DAPI) and anti-β-actin antibody were obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Recombinant human tumor necrosis factor-α (TNF-α) was obtained from R&D Systems (Minneapolis, MN, USA). BD Matrigel™ basement membrane matrix is supplied by BD Biosciences (San Diego, CA, USA).

The human breast cancer cell MDA-MB-231 was grown in RPMI-1640 supplemented with 10% FBS, 100 IU/ml penicillin and 10 μg/ml streptomycin. The human umbilical endothelial cell line EA.hy 926 was grown in DMEM supplemented with 10% FBS, 100 IU/ml penicillin and 10 μg/ml streptomycin. All cells were incubated in a humidified 5% CO₂ incubator.

Cells were seeded at 10⁴ cells per well in 24-well plates. After treatments, 50 μl of 5 mg/ml MTT solution was added to each well and incubated for 3 h. The supernatants were aspirated and the formazan crystals were dissolved with 200 μl of 4 N HCl-isopropanol in each well. The optical density of the colored product was measured at 570 nm, as suggested by the manufacturer, using an Infinite 200 microplate reader (Tecan Austria GmbH, Grödig, Austria).

Cells were seeded in 6-well plates at 1,000 cells/well. After serum starvation for 16 h, the cells were treated with morin at the indicated doses in a 37°C cell culture incubator. After 24 h, culture medium was discarded and changed with media every 2–3 days. After 1–2 weeks, cells were fixed and stained using crystal violet and photographed. The experiments were performed in triplicate.

Cells were washed in ice-cold PBS and lysed in PRO-PREP protein extraction solution (iNtRON Biotechnology, Seoul, Korea). The samples were centrifuged at 13,000 rpm, for 15 min at 4°C. An aliquot of the whole cell lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membrane. Membranes were blocked with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.05% Tween-20 for 2 h at room temperature and incubated with primary antibodies at 1:1,000 in TBS containing 0.05% Tween-20 and 3% bovine serum albumin overnight at 4°C. The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:5,000) antibody for 1 h at room temperature. After washing, the membranes were developed using the ECL reagent (Bionote, Gyeonggi-do, Korea).

The upper chamber of 24-well cell culture inserts (8-μm pore size, Falcon, Franklin Lakes, NJ, USA) were washed with a serum-free medium, coated with 100 μl of Matrigel (1 mg/ml) and dried for 30 min at 37°C. MDA-MB-231 cells treated with morin were collected; 2×10⁵ cells were loaded to the upper chambers filled with serum-free media, and 500 μl of RPMI media containing 10% FBS was added to the lower chambers. The invasion chambers were incubated for 24 h in a 37°C cell culture incubator. The non-invasive cells that remained on the upper surface of the insert membranes were removed by scrubbing. The cells on the lower insert membranes were stained with DAPI, and cells were counted under a fluorescence microscope. Each sample was measured in triplicate, and each experiment was repeated three times.

Gelatin zymography was performed as described by Jin et al (22). Briefly, conditioned media were concentrated using a protein concentrator (Thermo Pierce, Rockford, IL, USA) and subjected to electrophoresis on 8% PAGE gels containing 1 mg/ml gelatin. Gels were washed twice with 2.5% Triton X-100, stained with 0.2% Coomassie Brilliant Blue and destained (50% methanol and 10% acetic acid). Gelatinolytic activity was detected as clear bands in the background of blue staining.

Athymic nude mice were divided into 2 groups (5 mice/group) and received morin at the dose of 10 and 50 mg/kg (daily, i.p.), respectively for 7 days. Mice were injected subcutaneously with MDA-MB-231 (5×10⁶ cells/100 μl of serum-free RPMI). Tumors were allowed to grow until they reached 4 mm. At this point, mice were divided into 2 groups (7 mice/group): control and morin-treated mice. The mice were administered a daily i.p. injection of 10 mg/kg (non-toxic dose) morin for 45 days. The mice were sacrificed at day 45, and the tumors were extracted. Body weights and tumor volumes were measured every 3 days, starting at 7 days after injection. The experimental protocol was approved by the Institutional Animal Care and Use Committee at Gyeongsang National University.

Scanning densitometry was performed using Image Master^® VDS (Pharmacia Biotech Inc., San Francisco, CA, USA). The treatment groups were compared using one-way analysis of variance and the post hoc test by Scheffe. P<0.05 was considered statistically significant. All data were expressed as the mean ± standard error (SEM).

First, we examined the cell viability of MDA-MB-231 cells and normal endothelial cells in response to morin. When MDA-MB-231 cells and endothelial cells were treated with indicated doses of morin (10, 50, 100 and 200 μM) for 24 h, the results revealed that morin did not affect cell viability at the doses <100 μM both in MDA-MB-231 cells and endothelial cells. High concentrations (100 and 200 μM) of morin reduced cell viability of MDA-MB-231 a little but rather increased cell viability in endothelial cells (Fig. 1B). Thus, we investigated the effect of morin on the ability of MDA-MB-231 cells to form colonies. Interestingly, morin effectively inhibited colony formation of MDA-MB-231 cells in a dose-dependent manner (Fig. 1C).

Next, we observed changes of MDA-MB-231 cells in morphology after morin treatment. As shown in Fig. 2A, morin induced morphologic changes of MDA-MB-231 cells from mesenchymal form to epithelial form in a dose-dependent manner. We then determined the effects of morin on MDA-MB-231 cell invasion because cancer cell invasion is the first step for cancer metastasis. Matrigel invasion assays revealed that morin significantly inhibited cell invasion in a dose-dependent manner (Fig. 2B). Moreover, morin also effectively inhibited TNF-α-induced MDA-MB-231 cell invasion in a similar manner (Fig. 2C).

Proteolytic digestion of the extracellular matrix (ECM) by secreted MMPs is one of major steps for cancer invasion (23,24). MMP-2, -3 and -9 are also biomarkers for EMT (25). In particular, MMP-9 expression is associated with pathological processes, including inflammation, atherosclerosis and tumor-cell invasion and metastasis (26–28). Thus, we examined the effect of morin on the activity of the secreted MMP-9 from MDA-MB-231 cells in the presence of TNF-α or not by gelatin zymographic analysis. Morin dose-dependently suppressed the gelatinolytic activities of secreted MMP-9 in MDA-MB-231 cells. MMP-9 secretion augmented by TNF-α was also significantly inhibited by a low dose of morin (10 μM) (Fig. 3A). In addition, we also assessed the changes in EMT biomarkers to confirm that morin has inhibitory effects on EMT. Fig. 3B showed that morin inhibited mesenchymal markers N-cadherin, but did not influence the expressions of either vimentin or E-cadherin of MDA-MB-231 cells (data not shown). These results suggest that morin might suppress the invasion of highly metastatic MDA-MB-231 breast cancer cells through regulating the EMT process.

Concerning the upstream signals that affect N-cadherin, β-catenin induces N-cadherin expression, and β-catenin is negatively regulated by GSK-3β. GSK-3β is regulated by intracellular signaling pathways including PI3K/Akt. In other words, activation of PI3K/Akt results in the phosphorylation of GSK-3β (inactivation of GSK-3β), which in turn increases β-catenin protein levels. Thus, we investigated whether morin modulates Akt and GSK-3β pathways in MDA-MB-231. Morin reduced phosphorylated Akt level in MDA-MB-231 cells but failed to inhibit GSK-3β phosphorylation. In addition, TNF-α (10 ng/ml) enhanced Akt phosphorylation in MDA-MB-231, which was also significantly inhibited by morin. Morin did not reduce the TNF-α-mediated phosphorylation of GSK-3β (Fig. 4A and B). Then, we confirmed whether inhibition of Akt pathway could block the invasion of MDA-MB-231. As expected, inhibition of Akt pathway using LY294002 significantly decreased the invasion of MDA-MB-231 as well as TNF-α-enhanced invasion of MDA-MB-231 (Fig. 5A). In addition, LY294002 downregulated N-cadherin expression level (Fig. 5B). These results suggest that morin suppresses the invasion of MDA-MB-231 breast cancer cell through inhibiting Akt pathway and N-cadherin expression.

To confirm the in vivo effect of morin in tumor progression, morin was injected into tumor-bearing mice for 45 days. Before that, to determine whether morin has a toxic effect in vivo, the mice received i.p. a daily injection of 10 or 50 mg/kg morin for 7 days (n=7/group). Both 10 and 50 mg/kg concentrations of morin caused no lethality in mice (Fig. 6A), however 50 mg/kg made the mice somewhat nervous. Thus, we used 10 mg/kg of morin for further experiments. Control animals developed significant tumor growth during the 45-day follow-up period, as shown by the tumor volume in Fig. 6B and C. In contrast, animals that received 10 mg/kg morin daily showed that tumor growth was significantly inhibited (Fig. 6B and C). Body weight does not show any significant differences between the groups.