Ophiopogonin-D was isolated from the root of Ophiopogon japonicus according to the procedure of a previous study (27). Compounds were dissolved in dimethyl sulfoxide (DMSO; Nanjing Sunshine Biotechnology Ltd., Nanjing, China) and diluted with Dulbecco’s medium Essential medium (DMEM; Invitrogen Life Technologies, Carlsbad, CA, USA) without serum prior to each experiment. The final concentration of DMSO in the culture medium never exceeded 0.1% (v/v), a concentration known not to affect cell proliferation, and control groups were always treated with 0.1% DMSO in the corresponding experiments. MTT, Rose Bengal and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO, USA). High-glucose DMEM was purchased from Gibco-BRL (Invitrogen Life Technologies). Antibodies specific for phosphorylated (p-)ERK and p-p38/MAPK or total ERK, p38/MAPK, β-actin, MMP-2 and MMP-9 were obtained from Cell Signaling Technology (Cell Signaling Technology, Beverly, MA, USA). The cell culture plate and Transwell plate (8- μm pore size, 6.5-mm diameter) were obtained from Corning Costar Corporation (Corning, NY, USA). Matrigel was purchased from BD Biosciences (La Jolla, CA, USA).

The human breast carcinoma cell line MDA-MB-435 was obtained from Keygene Corporation (Nanjing, China), grown in high-glucose DMEM supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin and 3.7 g/l sodium bicarbonate (all purchased from Nanjing Sunshine Biotechnology Ltd.). HUVECs were obtained from Shanghai FuMeng Gene Biotechnology Co., Ltd. (Shanghai, China). The cells were maintained under a humidified atmosphere of 95% air and 5% CO₂ at 37°C and used for experiments whilst in logarithmic phase.

The tumor cell adhesion with HUVECs was detected according to the method by Jones et al (28) with certain modifications. HUVECs were seeded in 96-well plates in complete HUVEC medium at a density of 10⁴ cells per well for 24 h. The HUVEC medium was then removed and cells were washed with phosphate-buffered saline (PBS). MDA-MB-435 cells were trypsinized and suspended at a final concentration of 5×10⁵ cells/ml with various concentrations of ophiopogonin-D (5, 10, 20, 40 and 80 μM) dissolved in serum-free DMEM. After 3 h, the medium was removed and the undetached cells were washed with PBS twice and incubated with Rose Bengal for 5 min. The cells were washed with PBS twice and fixed with 100 μl mixed solution (95% ethanol/PBS 1:1) per well for 30 min at room temperature. The ratio of adhesion was detected by measuring the absorbance of Rose Bengal at a wavelength of 600 nm using an ELISA reader (Bio-Tek Instruments, Winooski, VT, USA).

Cell adhesion to fibronectin was assayed as described previously with a few alterations (29). Briefly, 96-well plates were coated with fibronectin (Merck, Whitehouse Station, NJ, USA) at 4°C overnight. MDA-MB-435 cells, which were trypsinized and suspended at a final concentration of 5×10⁵ cells/ml in serum-free DMEM with various concentrations of ophiopogonin-D and cultured for 3 h. The unattached cells were removed by discarding the media and washing with PBS twice. A total of 100 μl MTT (0.5 mg/ml in PBS) was added to the wells and the cells were cultured for another 3 h. Following discarding the MMT solution, DMSO was then added to the wells. After agitation for 10 min at room temperature, the cytotoxicity was determined by measuring the absorbance of the converted dye at a wavelength of 570 nm and a reference wavelength of 650 nm in an ELISA reader.

The cell invasion assay was determined as described previously with a few modifications (30). Briefly, the Transwell membrane (8 μm pore size, 6.5 mm diameter; Corning Costar Corporation) was pre-coated with Matrigel. The MDA-MB-435 cells, which were trypsinized and suspended at a final concentration of 5×10⁵ cells/ml in serum-free DMEM with various concentrations of ophiopogonin-D, were added to the upper well, while 500 μl DMEM containing 10% FBS was added to the lower well. After 20 h, assays were stopped by removal of the medium from the upper wells. The cells on the membrane were fixed with 90% ethanol and cells on the upper side of the membrane, which had not transgressed through the membrane, were subsequently wiped off using cotton buds. The cells on the lower side of the membrane were stained with 0.1% methyl violet. After 10 min, the chamber was washed with PBS and images of the membrane were captured using a microscope (Axiovert40CFL; Carl Zeiss, Oberkochen, Germany). Finally, the chamber was extracted with 10% acetic acid, and the absorbance of the extract was measured at 570 nm in an ELISA reader.

The activities of MMP-2 and MMP-9 were assayed by gelatin zymography (31,32). Gelatin zymography was performed in 10% SDS-polyacrylamide gel containing 0.1% gelatin. After treatment with various concentrations of ophiopogonin-D (5, 10, 20, 40 and 80 μM) for 24 h, the supernatants were collected. Samples were mixed with loading buffer and electrophoresed. Gels were then washed twice in zymography washing buffer (2.5% Triton X-100, 50 mmol/l Tris-HCl, 5 mmol/l CaCl₂; pH 7.6) for 45 min at room temperature to remove SDS, followed by rinsing twice in washing buffer for 20 min. The gels were then incubated at 37°C for 40 h in zymography reaction buffer (50 mmol/l Tris, 150 mmol/l NaCl,10 mmol/l CaCl₂, 0.02% NaN3, pH 7.5) and stained with 0.05% Coomassie blue R-250 (diluted in 30% methanol and 10% acetic acid) for 3 h and destained with destaining solution (10% methanol and 5% acetic acid in H₂O₂). The field of the destained band was quantified using the Quantity One System (Bio-Rad, Hercules, CA, USA).

Activation of MMP-2/9 and MAPK were assessed by western blot analysis. MDA-MB-435 cells were pre-incubated with various concentrations of ophiopogonin-D for 20 h, and cells were collected. Cells were then lysed in lysis buffer, and the equal amount of proteins was separated by SDS-PAGE and transferred onto the polyvinylidene difluoride membranes. Membranes were blocked with blocking buffer for 1.5 h at room temperature, followed by overnight incubation at 4°C in the relevant primary antibody (1:1,000), and finally blocked for 1 h with a secondary antibody HRP-conjugate (1:2,000). The band detection was revealed by enhanced chemiluminescence (ECL) using ECL western blotting detection reagents (Vazyme Biotech Co., Ltd., Nanjing, China) and exposure to ECL hyperfilm on Kodak film (Eastman Kodak, Rochester, NY, USA) and analyzed using the Quantity One System.

Values are expressed as the mean ± standard deviation. The experimental data were analyzed using GraphPad Prism 5 software (GraphPad Inc., La Jolla, CA, USA). All comparisons were made relative to the control groups and significance of differences was indicated as ^*P<0.05 and ^**P<0.01.

In a preliminary experiment, the cytotoxic effects of ophiopogonin-D were determined using an MTT assay (data not shown). Ophiopogonin-D demonstrated no obvious cytotoxicity on MDA-MB-435 cells under 80 μM. Concentrations of ophiopogonin-D (5, 10, 20, 40 and 80 μM) with no apparent cytotoxicity on MDA-MB-435 cells were selected for all subsequent experiments. As shown in Fig. 2A, ophiopogonin-D was able to inhibit the adhesion of MDA-MB-435 cells to HUVECs. Ophiopogonin-D at concentrations of 40 and 80 μM significantly reduced the number of MDA-MB-435 cells adhered to HUVECs, and the inhibition rates were ~50 and 65%, respectively. In addition, MDA-MB-435-cell adhesion to fibronectin was detected for more accurate determination of the effect of ophiopogonin-D on the adhesion of melanoma cells. Fig. 2B shows the inhibitory effect of ophiopogonin-D on the adhesion of MDA-MB-435 cells to fibronectin. At concentrations of 40 and 80 μM, ophiopogonin-D significantly inhibited the adhesion of MDA-MB-435 cells to fibronectin at rates of ~64 and 80%, respectively. Furthermore, the effect of ophiopogonin-D on the invasive ability of MDA-MB-435 cells was evaluated using a Transwell assay. Fig. 2C indicates that after treatment with ophiopogonin-D the invasive capacity of MDA-MB-435 cells was markedly inhibited. The inhibition rate of 80 μM of ophiopogonin-D was ~78%.

The expression of MMP-2 and MMP-9 has been reported to have a critical role in degrading the basement membrane in tumor invasion and migration (33). A gelatin zymography assay showed that the activity of MMP-9 was reduced with increasing concentration of ophiopogonin-D (Fig. 3). MMP-9 was obviously decreased following incubation with 40 and 80 μM ophiopogonin-D, while there was no obvious decrease in MMP-2. This result indicated that ophiopogonin-D mostly inhibits tumor metastasis through MMP-9, not MMP-2.

MMPs have significant roles in cancer metastasis. Furthermore, the activity of MMP-2/9 is associated with the adhesion, invasion and angiogenesis of cancer metastasis (34,35). Therefore, the effect of ophiopogonin-D on the expression of MMP-2 and MMP-9 was deteced by western blot analysis. The results indicated that ophiopogonin-D inhibited the expression of MMP-9 at concentrations of 40 and 80 μM (Fig. 4). With increasing ophiopogonin-D concentration, the expression of MMP-9 declined, while there was no obvious decrease in MMP-2 expression. The result of the western blot analysis corresponded with the results of the adhesion and invasion experiments. Furthermore, the expression and phosphorylation of ERK1/2 and p38/MAPK were examined to assess whether the MAPK signaling pathway was involved in the anti-metastatic effect of ophiopogonin-D in melanoma cells (Fig. 4). The phosphorylation of p38 was inhibited by ophiopogonin-D at the concentration of 80 μM, while the phosphorylation of ERK was not inhibited by ophiopogonin-D. Further investigation is required to fully elucidate the underlying mechanism of the anti-metastatic effect of ophiopogonin-D on melanoma cells.