Human fibrosarcoma HT1080 cells were obtained from the Korean cell line bank (KCLB no. 10121, Seoul, Korea). The cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 (Gibco-Invitrogen, Carlsbad, CA, USA) medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 U/ml streptomycin in a humid atmosphere of 5% CO₂ and 95% air at 37°C.

The HT1080 cells were plated into 96-well plates (1×10⁴ cells/well for cells in 100 μl of medium). After an overnight incubation, the cells were treated with gradient concentrations of SAL (0, 1, 2, or 5 μM) for 24 h or with 5 μM SAL for the indicated time periods, and then washed with phosphate-buffered saline (PBS). Thereafter, the medium was replaced with fresh medium (200 μl) containing 0.5 mg/ml MTT, and the mixture was incubated for 4 h at 37°C. Following incubation, 150 μl of dimethyl sulfoxide (DMSO) was added to each well to terminate the MTT reaction and to dissolve the formazan crystals. Subsequent to agitation for 1 h at room temperature, the optical density of the cells in each well was measured at 570 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Percentage of cell viability was calculated based on untreated HT1080 (control) cells assigned 100% viability.

For detection of HT1080 cell migration, a scratch assay of the HT1080 cells was performed. In total, 2×10⁵ cells/well HT1080 cells were seeded into 35-mm culture dishes. The cells were cultured overnight and scraped using a 10-μl pipette tip to create a straight-line scratch. The remaining cells were washed with medium and incubated with SAL in the absence or presence of inhibitors for 24 h. Images of the scratch were then captured at 24 h, and the images were quantitatively analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA). The distance between the edges of the scratch were measured and statistically analyzed.

The invasive ability of HT1080 cells was detected using the Transwell system, which consisted of 24-wells, an 8-μm pore size Transwell filter, and a polycarbonate membrane coated with Matrigel (Corning Inc., Corning, NY, USA). Initially, serum-free Dulbecco's modified Eagle's medium (DMEM) diluted Matrigel matrix (60 μg/well) was put into the upper chamber of the Transwell filter and incubated for 4 h at 37°C for gelling. Subsequently, the cells were trypsinized, and 2×10⁵ cells in 100 μl serum-free medium with or without SAL were added to each upper chamber. Following incubation for 24 h, the cells attached to the upper surface of the membrane were removed with cotton swabs. Cells that had migrated to the underside of the insert membrane were fixed with methanol and dried. The cells were then stained for 20 min with 1 mg/ml crystal violet, rinsed in deionized water, air-dried, and observed under a microscope (Olympus BX50 microscope, Tokyo, Japan). At least 20 random fields were counted per filter. Each experiment was performed in triplicate and repeated at least twice.

In total, 2×10⁵ cells/well were seeded into 35-mm culture dishes and starved in serum-free medium at 37°C. Subsequent to a 12-h starvation period, the cells were treated with SAL. The cells were then trypsinized and washed with PBS prior to being suspended in cold propidium iodide (PI) solution (50 μg/ml) containing RNase A (0.1 mg/ml) in PBS (pH 7.4) for 30 min in the dark. The DNA content of the cells was analyzed using a FACScaliber flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and the data were analyzed by using BD FACStation software (BD Biosciences).

The cells were collected and the total cell lysates were prepared using cold radio immuno-precipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% nonidet P-40, and 0.1% sodium dodecyl sulfate (SDS) supplemented with protease inhibitors (10 μg/ml leupeptin, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, and 1 mM of 4-[2-aminoethyl] benzenesulphonyl fluoride) and phosphatase inhibitors (1 mM NaF and 1 mM Na₃VO₄). Cell lysate was centrifuged at 13,000 rpm for 10 min at 4°C. The protein concentration was then measured according to the bicinchoninic acid (BCA) method, with bovine serum albumin (BSA) as the standard. Next, 20–40 μg equal amounts of whole cell lysates were resolved on 8–12% SDS-polyacrylamide gel electrophoresis (PAGE) at constant voltage and then transferred to a nitrocellulose membranes. The membranes were incubated in blocking buffer composed of 5% skim milk in Tris-buffered saline with Tween-20 (TBST) at room temperature for 1 h. Next, the blocked membranes were incubated overnight with primary antibodies at 4°C. Thereafter, the membranes were washed with TBST three times for 10 min each and then incubated with secondary antibodies at room temperature for 2 h. The blots were also incubated with a polyclonal mouse anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody, which acted as an internal control for the quantity of target protein. Subsequent to washing in TBST, immunoreactive bands were visualized under image analyzer (LAS-4000, FujiFilm, Tokyo, Japan) using an enhanced chemiluminescence kit (Dogen, Seoul, Korea).

Gelatinase activity in the conditioned media was determined by gelatin zymography, as previously described (18). Cell conditioned medium was electrophoresed using 7.5% SDS-PAGE containing 0.1% (w/v) gelatin under non-reducing conditions and without boiling. Gels were washed twice with 2.5% (v/v) triton X-100 for 20 min at room temperature and subsequently incubated overnight at 37°C in a developing buffer containing 10 mM CaCl₂, 150 mm NaCl, and 50 mM Tris-HCl, pH 8.0. The gels were then stained with 0.5% Coomassie Brilliant Blue R-250 in 10% acetic acid (v/v) and 50% methanol. The gels were destained with 50% methanol and 10% acetic acid solution, scanned, and subjected to densitometry analysis using ImageJ software.

Total cellular RNA was extracted from treated cells using TRIzol reagent (Gibco). Total RNA (0.5 μg) was used for cDNA synthesis with the maxime RT-PCR Premix kit (Intron, Seongnam, Republic of Korea). PCR was carried out in Takara thermal cycler dice (Takara Bio, Inc., Shiga, Japan) to amplify MMP-2 and GAPDH mRNA. Primer sequences used to amplify the desired cDNA were as follows: MMP-2 (forward primer: GCC TGA GCT CCC GGA AAA GAT TG, reverse primer: CAG CAG CCT AGC CAG TCG GAT TT), GAPDH (forward primer: CGT CTT CAC CAC CAT GGA GA, reverse primer: CGG CCA TCA CGC CAC AGT TT). PCR products electrophoresed on 2% agarose gels were visualized by ethidium bromide staining.

Data are presented as means ± standard error of the mean (SEM) [means ± standard deviation (SD) in the results] from at least three independent experiments and evaluated by analysis of variance (ANOVA) followed by Tukey's post-hoc test. Values of p<0.05 were considered statistically significant.

To determine the effect of SAL on HT1080 cells, an MTT assay was performed to measure cellular proliferation (Fig. 1). HT1080 cells were treated with the various concentrations of SAL for 24 h (Fig. 1). As shown in Fig. 1A, the proliferation of cells treated with SAL was significantly lower than that of the control cells. SAL suppressed proliferation in a dose- and time-dependent manner (Fig. 1). Flow cytometry was also used to determine the ability of SAL to induce cell cycle arrest or affect the cell cycle distribution of HT1080 cells (Fig. 1B). Compared to control cells, cells treated with SAL exhibited increased G₂/M arrest at ~5 μM, which was caused by a decrease in the ratio of S phase cells and an increase in the ratio of subG1 phase cells at 10 μM (Fig. 1B). Next, HT1080 cells were treated with 1, 2, or 5 μM SAL to study the effect of SAL on the migration and invasion on these cells. We found that SAL decreased proliferation in a dose-dependent manner, with cytotoxic effects identified at 10 μM SAL, indicating an inhibitory effect of SAL on cell proliferation. Therefore, all of the obtained data were corrected by the ratio of proliferation.

Since MMP-2 and MMP-9 play a pivotal role in tumor cell invasiveness, we determined the effect of SAL on MMP-2 and MMP-9 enzyme activities. Gelatin zymography and western blot analysis were performed in order to investigate the effect of SAL on MMP-2/-9 activity and expression (Fig. 2). We confirmed that SAL induced MMP-2 expression in a dose- and time-dependent manner, as determined by western blot analysis (Fig. 2A). However, SAL did not affect activity or the expression of MMP-9 enzyme (data not shown). The mRNA level of MMP-2 was measured by quantitative RT-PCR (Fig. 2B). SAL treatment increased MMP-2 mRNA within 3 h (Fig. 2B, lower panel).

The results from gelatin zymography were consistent with the results found in western blot analysis, as described above. SAL significantly induced MMP-2 activity in a dose- and time-dependent manner. The active bands of MMP-2 gradually increased when cells were treated with increasing concentrations of SAL (from 1 to 5 μM, Fig. 2C).

To determine whether SAL affected the motility of HT1080 cells, migration and scratch assays were performed (Fig. 3). Confluent monolayers of cells were wounded with a uniform scratch, washed to remove debris, and incubated in the absence or presence of SAL for 24 h (Fig. 3A). The result of the scratch assay revealed that the wound closure level, which corresponds to the wound healing ability, was significantly increased in a dose-dependent manner compared with the untreated control cells (Fig. 3A). The invasion assay showed that the number of cells that had invaded through the Matrigel-coated polycarbonate membrane was significantly increased in SAL treated cells compared to that of the control cells. These data were corrected by the ratio of proliferation (Fig. 3B). The results indicated that SAL increased the invasion and migratory capacity of HT1080 cells (Fig. 3).

Several studies have indicated that the PI-3 kinase and MAPK pathways are involved in regulating the activity of MMP-2 in various cell types. To assess whether SAL regulates PI-3 kinase and MAPK pathways, we investigated the phosphorylation status of PI-3 kinase/Akt and MAPKs (ERK-1/2, p38, and JNK) in HT1080 cells after treatment with various concentrations of SAL for 24 h (Fig. 4A) or 5 μM SAL at different time periods (Fig. 4B). As shown in Fig. 4, SAL significantly increased the phosphorylation of PI-3 kinase/Akt and the MAPKs, ERK-1/2 and p38 kinase (Fig. 4). Phosphorylated JNK was not detected in SAL-treated cells (data not shown).

To verify that the PI-3 kinase and MAPKs pathway are involved in SAL induced migration and invasion, cells were treated with PI-3 kinase and MAPK pathway inhibitors in addition to SAL (Fig. 5), and inhibition of PI-3 kinase and MAPK pathways with LY294002 (LY), PD98059 (PD), or SB203580 (SB) abolished SAL-induced MMP-2 expression, migration, and invasion (Fig. 5). Taken together, our data suggest that the PI-3 kinase and MAPK (ERK-1/2, and p38 kinase) pathways are involved in SAL-induced MMP-2 expression, migration, and invasion in HT-1080 cells.