Loquat leaves were collected from the Herb Garden of Mokpo National University in Muan, Korea. Leaves were freeze-dried and powdered. Loquat seeds were collected from Wan-Do in Jeonnam, Korea and used for preparation of extract after peeling of hard shells. The methanol extracts were obtained by macerating leaf powder and seeds with 80% methanol for 2 days at room temperature, and this procedure was repeated twice. The respective extract was filtered under reduced pressure and freeze-dried. The yields obtained were 32% for leaves and 27% for seeds.

The MDA-MB-231 human breast cancer cells were obtained from the American Type Culture and Collection (Rockville, MD). All cells were grown in Dulbecco's modified Eagle's medium (DMEM; Gibco, Gaithersburg), supplemented with penicillin 100units/ml, streptomycin 100 µg/ml and 10% fetal bovine serum, and incubated in a humidified incubator at 37℃ and 5% CO2. Cultures used in subsequent experiments were at < 20 passages.

To determine the effect of leaf and seed methanol extracts on MDA-MB-231 cell proliferation, cells were seeded in 6-well culture plates at a concentration of 6×10⁴/well. Complete medium with DMSO or with 10, 25 or 50 µg/ml of leaf or seed extract were replaced every other day, beginning the day after seeding. Cells cultured in complete medium were harvested with trypsin-EDTA on day 5 after treatment and counted by a model Z1 Coulter particle counter (Coulter Corp, Miami, Florida).

Plates (12-wells) were coated with 30 µg of Matrigel (Becton Dickinson, Bedford, MA). To block nonspecific binding sites, Matrigel coated culture plates were re-hydrated with serum free DMEM containing 0.1% BSA for 90 min at 37℃ and then washed with the same medium. MDA-MB-231 cells were trypsinized and resuspended in serum free DMEM containing 0.1% BSA. Cells were seeded at a concentration of 2×10⁵/well in the presence of DMSO or of 10, 25 or 50 µg/ml of leaf or seed extract and incubated for 90 min at 37℃ and 5% CO₂. At the end of 90 min, the medium was removed and the cells were washed gently two times with PBS to remove unattached cells. The attached cells were harvested by trypsinization, resuspended and counted. Each assay was performed in triplicate and repeated in three independent experiments. Values were expressed as the average of triplicate experiment.

Motility was assessed using a scratch wound assay (Goodman et al., 1985). Cells were seeded into 6-well cell culture plates at a concentration of 3×10⁵cells and cultured in 10% FBS DMEM to near confluence. The confluent monolayer was carefully wounded using a sterile pipette tip and cellular debris was washed gently with PBS. The wounded monolayer was incubated in 10% FBS DMEM containing 20 µg/mL fibronectin and DMSO or 10, 25 or 50 µg/ml of leaf or seed extract for 24 hours. Migrating cells were examined under 10× by phase contrast microscope and photographed.

The ability of cells to migrate across a Matrigel barrier (invasion) was determined by a modified Boyden chamber. Briefly, Boyden chambers were assembled using 8 µm Falcon transwell inserts (Becton Dikinson, Bedford, MA) as the upper chamber and 12-well plates as the lower chamber. Matrigel was applied to the insert and left overnight to dry in a laminar flow fume hood. The insert was re-hydrated with serum free DMEM for 90 min at 37℃ and 5% CO₂. Following rehydration, Matrigel coated inserts were washed with serum free DMEM. Exponentially growing breast tumor cells were harvested by trypsin/EDTA and resuspended in 0.1% BSA DMEM. Cells were seeded at a concentration of 2×10⁵ cells. DMSO or 10, 25 or 50 µg/ml of leaf or seed extract was carefully added to the upper chamber. Twenty micrograms of fibronectin was added as a chemoattractant to the lower chamber. The Boyden chamber was incubated for 24 hours at 37℃ and 5% CO₂. At the end of incubation, the cells in the upper chamber were removed by wiping gently with a cotton swab. Cells that transversed the Matrigel and attached to the lower surface of the insert were fixed with 10% formalin and stained with 0.5% crystal violet. Inserts were examined under 20× or 40× phase contrast field microscopy and photographed. Values for invasion were expressed as the number of migrated cells per microscopic field. Four fields were counted. Each assay was performed in triplicate and repeated in three independent experiments. Values were expressed as the average of triplicate experiments.

The activity of MMP-2 and MMP-9 in the conditioned media was assayed by gelatin zymography. Briefly, MDA-MB-231 cells were seeded at a concentration of 2×10⁵/well in 6-well plates. After treatment with DMSO or 10, 25 or 50 µg/ml of leaf or seed extract for 24 hours, the supernatants were collected and subjected to gel electrophoresis on 10% running gels containing 0.1% gelatin. The gels were washed in 2.5% Triton X-100 for 30 minutes, followed by incubation for 20 hours at 37℃ in the incubation buffer containing 50 mM Tris-HCl (pH 7.5), 0.2 M NaCl, 1 mM CaCl₂ and 0.2% NaN₃. The gels were stained for 10 hours in 0.5% Coomassie brilliant blue, then destained with destaining solution (45% methanol and 10% acetic acid) and photographed.

Statistics were analyzed using Sigma Stat software. Results were expressed as the mean ± S.D. of three independent experiments. Comparisons were based on one-way ANOVA followed by Duncan's multiple range test. A p-value < 0.05 was considered statistically significant.

Loquat leaf extract inhibited the proliferation of MDA-MB-231 cells at all concentrations and was more effective than seed extract (Fig. 1). Seed extract significantly inhibited proliferation at the concentrations of 25 and 50 µg/ml, but not at 10 µg/ml. At the concentration above 100 µg/ml of loquat leaf or seed extract, the number of cells on the day 5 after treatment was decreased to less than 6×10⁴/well, the number of cells seeded at the beginning of proliferation assay. However, extracts at the concentrations of 10, 25, and 50 µg/ml inhibited the cell growth and no cytotoxicity was observed. Thus, we used extracts at the concentrations of 10, 25, and 50 µg/ml for further investigation of anti-metastatic effect of loquat.

Loquat inhibited the adhesion of cancer cells. Leaf extracts down-regulated the binding of the cells to Matrigel and significantly inhibited adhesion of MDA-MB-231 cells in a dose dependant manner (Fig. 2). At a concentration of 50 µg/ml, adhesion was reduced by 56% compared to the control. Seeds had a significant inhibitory effect on adhesion only at a concentration of 50 µg/ml.

The effect of loquat on MDA-MB-231 cell migration was analyzed using the scratch wound assay. In the absence of loquat extract, MDA-MB-231 cells migrated along the edges of the wound and covered the wound. Significant inhibition of cell flattening and spreading was observed in the presence of loquat leaf and seed extracts (Fig. 3).

We analyzed the invasion of MDA-MB-231 using a Boyden chamber model, and both leaf and seed extract dose dependently suppressed the invasion of MDA-MB-231cells (Fig. 4). Loquat leaf extract inhibited the invasion of breast cancer cells more effectively than seed extract.

We analyzed the effect of loquat on the activities of gelatinolytic MMP-2 and MMP-9 in MDA-MB-231 breast cancer cells to determine whether the anti-invasive activity of loquat was correlated with the inhibition of MMP activity (Fig. 5). Loquat leaf extract had an inhibitory effect on the MMP-9 activity in a dose dependent manner, and MMP-2 activity only at the concentration of 50 µg/ml. Seed extract showed an inhibitory effect on the MMP-9 activity.