All plastic materials were purchased from Falcon Labware (Becton Dickinson and Company, Franklin Lakes, NJ, USA). Fetal bovine serum (FBS), phosphate-buffered saline and penicillin G/streptomycin were obtained from Gibco-BRL (Grand Island, NY, USA). RPMI-1640 medium was purchased from Welgene, Inc. (Daegu, Korea). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. (Gaithersburg, MD, USA).

SCL was purchased as a dried herb from Omniherb Co. (Yeoungcheon, Korea). The dried bark of SCL (200 g) was extracted twice using 70% ethanol with 2 h reflux, and the extract was concentrated under reduced pressure. The decoction was filtered, lyophilized and stored at 4°C. The yield of dried extract from the starting crude material was ~12.7% (w/w). The SCLE was diluted in culture media to the final concentrations indicated for each experiment.

MDA-MB-231 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. The culture was performed at 37°C in 5% CO₂. Subsequent to reaching 80% confluence, the cells were detached using 0.25% trypsin-EDTA. Cells were then subcultured further.

Water-soluble tetrazolium salt-8, which is reduced to yellow water-soluble formazan by the dehydrogenase released from mitochondria, was the effective constituent of the CCK-8. Absorbance was measured at 450 nm using a Benchmark Plus Microplate Spectrophotometer (Bio-Rad, Hercules, CA, USA). Cytotoxicity was expressed as a percentage of the absorbance measured in the control and SCLE-treated cells.

MDA-MB-231 cells were plated in a 12-well plate at a concentration of 5×10⁵ cells/well and allowed to form a confluent monolayer for 24 h. Cells were wounded using a yellow pipette tip and cellular debris was removed by washing with RPMI-1640 medium. The wounded monolayer was incubated in the presence or absence of SCLE (25 and 50 μg/ml) for 24 h in either RPMI-1640 plus 10% FBS as a positive control or RPMI-1640 plus 1% FBS (CON). Images were captured of the cell migration into the wounded area using phase-contrast microscopy.

qPCR was performed using the Rotor-Gene 3000 (Corbett Research, Sydney, Australia) system using SYBR^® Green Master Mix (Qiagen, Tokyo, Japan). The percentage of target gene expression relative to the control was normalized using the GAPDH internal control according to the 2^−ΔΔCt analysis. Primers for the target genes were designed using Primer3 software (Table I) (14).

Statistical analyses were performed using the Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). Analysis of variance followed by a Dunett’s test were performed. A value of P<0.05 was considered to indicate a statistically significant difference.

The cytotoxic effect of SCLE was investigated in three cancer cell lines (HepG2, A549 and MDA-MB-231) using the CCK-8. SCLE was observed to reduce the viability of each of these cell lines in a concentration-dependent manner (Fig. 1A). However, SCLE was found to have a weaker effect on the viability of A549 and MDA-MB-231 cells than that of HepG2 cells. SCLE was observed to reduce the viability of MDA-MB-231 cells from 100 to 60% in a concentration- and time-dependent manner (Fig. 1B). The viability of MDA-MB-231 cells was significantly reduced following treatment with 100–400 μg/ml SCLE for 12 or 24 h (P<0.01) (Fig. 1B). Based on these results, non-cytotoxic concentrations of SCLE (25 and 50 μg/ml) were used in further experiments.

Due to the highly invasive nature of triple-negative breast cancers, the present study investigated the effect of SCLE on the invasive potential of MDA-MB-231 cells in vitro. A wound healing assay was performed to determine the effect of SCLE on the inhibition of MDA-MB-231 cell migration. Twenty-four hours after cell monolayers were wounded, control cell monolayers (RPMI-1640 plus 1% FBS) had completely filled the wounded area. SCLE treatment was observed to inhibit the migration of MDA-MB-231 cells in a concentration-dependent manner (Fig. 2A). Densitometric analysis showed that treatment with 50 μg/ml SCLE reduced the migration of MDA-MB-231 cells by ~50% (Fig. 2B). These results suggest that SCLE effectively inhibits the migration of the highly invasive MDA-MB-231 breast cancer cell line.

The overexpression and activation of urokinase plasminogen activator (uPA) and the uPA receptor (uPAR) play an important role in the invasion of breast cancer cells by stimulating ECM degradation and thereby promoting cell migration. Therefore, the present study aimed to investigate whether the SCLE-induced inhibition of migration in MDA-MB-231 cells correlated with modulation of uPA and uPAR. SCLE treatment was found to reduce the mRNA levels of uPA and uPAR in a concentration-dependent manner (Fig. 3). By contrast, the mRNA levels of tissue inhibitor of metalloproteinase (TIMP) 1 and 2 were significantly increased following SLCE treatment (P<0.05). These data suggest that the SCLE-mediated inhibition of MDA-MB-231 cell migration and metastasis may be dependent on the degradation of the ECM.