The brown alga S. thunbergii was collected from the coast of Jeju Island, Korea. The HT1080 cell line was obtained from the American Type Culture Collection. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin/amphotericin (10,000 U/ml, 10,000 μg/ml, and 2,500 μg/ml, respectively), phosphate-buffered saline (PBS), and 0.25% trypsin-ethyl-enediaminetetraacetic acid (EDTA) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), gelatin (type A), PMA, dimethyl sulfoxide (DMSO) and silica gel were purchased from Sigma-Aldrich (Merck KGaA).

The specific anti-MMP-9 rabbit polyclonal antibody (ab38898) was purchased from Abcam. Rabbit polyclonal antibodies against extracellular signal-regulated kinase (ERK) 1/2 (sc-292838), p38 (sc-7149), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778), mouse monoclonal antibodies against phosphorylated (p)-ERK 1/2 (sc-7383), c-Jun N-terminal kinase (JNK) 1/2 (sc-7345), p-JNK 1/2 (sc-6254), p-p38 (sc-7973), and nuclear factor-κB (NF-κB) p65 (sc-8008), and horseradish peroxidase-conjugated donkey polyclonal secondary antibody against goat IgG (sc-2020) and goat poly-clonal secondary antibody against mouse IgG (sc-2031) were all purchased from Santa Cruz Biotechnology, Inc. The anti-inhibitor of NF-κB α (IκBα) rabbit polyclonal (cat. no. 9242) and anti-p-IκBα rabbit monoclonal (cat. no. 2859) antibodies were purchased from Cell Signaling Technology, Inc. The Alexa Fluor^® 546-conjugated goat polyclonal anti-mouse IgG (H+L) cross-absorbed secondary antibody (A-11030), Alexa Fluor 633-conjugated goat polyclonal anti-rabbit IgG (H+L) cross-adsorbed secondary antibody (A-21070), and Hoechst 33342 nucleic acid stain (H1399) were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). Coomassie Brilliant Blue R-250 was purchased Biosesang. Other common analytical-grade chemicals and reagents used in this study were commercially available.

After collection, all algal samples were washed three times with tap water to remove salt, sand and epiphytes attached to the surface, and were carefully rinsed again with fresh water. The samples were stored at -20°C in a medical refrigerator until use.

The frozen algae were lyophilized and homogenized into powder. The powdered S. thumbergii (2 kg) was extracted three times with 80% methanol, and the methanol extract was centrifuged at 3,500 x g for 30 min at 4°C and then filtered with Whatman No. 1 (Whatman Ltd.) filter paper to remove the residue. The filtrate was evaporated at 40°C to obtain a methanol extract, which was then suspended in distilled water (DW) and partitioned using chloroform. The chloroform fraction (2 g) was subjected to silica column chromatography by stepwise elution with a chloroform-methanol solution (30:1→1:1, v/v) to separate the active fraction (12.3 mg) in chloroform fraction. Silica gel (230-400 mesh) was packed in a glass column (Pyrex, 300x38 mm, Corning Inc.) and a flow of solvent by gravity was allowed during the purification.

The active fractions were further separated by a Sephadex LH-20 column saturated with 100% methanol. Next, the active compounds were purified by reversed-phase high performance liquid chromatography (HPLC) using a Waters HPLC system (Alliance 2690; Waters Corporation) equipped with a Waters 996 photodiode array detector and a C18 column (J'sphere ODSH80, 250x4.6 mm, 4 μm; YMC) by stepwise elution with methanol-water gradient (ultraviolet absorbance detection wavelength, 296 nm; flow rate, 1 ml/min; injected volume, 30 μl). The HPLC eluting conditions were as follows: 5-70% methanol for 40 min to 100% methanol for 20 min, followed by 10 min re-equilibration time of the column. HPLC anlaysis was performed in a room kept at 24±1°C.

Finally, the purified compound was identified by comparing their 1H- and 13C-nuclear magnetic resonance (NMR) spectra with reported data (36). NMR spectra (data not shown) were recorded on a ZEOL 600 MHz NMR spectrometer (JEOL). The chemical structure of I6CA isolated from S. thunbergii is shown in Fig. 1. For cell culture experiments, I6CA was dissolved in DMSO, and the final concentration of DMSO in culture medium was adjusted to ~0.01%.

HT1080 cells were routinely cultured in complex medium (DMEM supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin/amphotericin). Cultures were maintained at 37°C, 5% CO₂ in a humidified atmosphere. The cells were subcultured every 3 days using trypsin-EDTA for 3 min for cell detachment.

HT1080 cells were seeded in a 96-well plate at a density of 104 cells/well in 100 μl of complete medium. The next day, HT1080 cells were treated, in the absence or presence of serum, with increasing concentrations of I6CA (100, 200 and 400 μM) in the presence or absence of PMA (10 ng/ml) at 37°C. After 24 h of incubation, 20 μl of MTT stock solution (1 mg/ml in PBS) was added to each well. After 2 h of incubation at 37°C, the supernatant was removed, and in each well, the formazan crystals were dissolved in 100 μl of DMSO. Then, the absorbance was measured at 540 nm using a Powerwave XS2 microplate reader (BioTek Instruments, Inc.). The relative cell viability was calculated based on the quantity of MTT converted to formazan by the untreated and PMA only-treated cells (100% viability). The data are expressed as the mean percentage of the viable cells ± standard deviation of triplicate experiments.

The gelatinolytic activity of MMP-9, secreted from HT1080 cells, was determined by gelatin zymography (37-39). HT1080 cells grown in 6-well plate at a density of 105 cells/well in 2 ml of complete medium were treated with I6CA (100, 200 and 400 μM) in serum-free medium in the presence or absence of PMA (10 ng/ml) for 24 h at 37°C. Cell culture supernatants were collected and their protein contents were measured using a bicinchoninic assay (BCA) protein assay kit (Thermo Fisher Scientific, Inc.) with a standard curve of a range of bovine serum albumin concentrations in DW (0-1 mg/ml; Thermo Fisher Scientific, Inc.). 20 μl of cell culture supernatants were subjected to electrophoresis on 10% SDS-polyacrylamide gels containing 0.25% gelatin. The gel was washed in 2.5% Triton X-100 at room temperature to remove SDS and then incubated overnight at 37°C in developing buffer (50 mM Tris-HCl at pH 7.5, 200 mM NaCl, 5 mM CaCl₂·2H₂O, and 0.02% Brij-35). Finally, the gel was stained at room temperature with Coomassie Blue staining solution (1% Coomassie Brilliant Blue R-250, 45% methanol, and 10% acetic acid) for 30 min and destained using the same solution without dye. Negative staining (clear bands against a blue background) indicated proteolysis and, therefore, gelatinolytic activity. The MMP-9 gelatinolytic activity was quantified by measuring the band intensities using ImageJ 1.8.0. software (National Institutes of Health). The normalization of loading protein was conducted via electrophoresis with 10% SDS-polyacrylamide gels after which Coomassie staining to analyze the amount of total loading protein.

HT1080 cells were lysed for 30 min in lysis buffer (20 mM Tris-HCl at pH 7.4, 5 mM EDTA, 10 mM Na₄P₂O₇, 100 mM NaF, 2 mM Na₃VO₄, 1% NP-40, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 1 mM PMSF) and then cell debris was removed by centrifugation for 15 min at 16,000 x g, 4°C. The protein concentration of the cell lysates was determined using a BCA protein assay kit. Next, 30 μg of proteins were separated by electrophoresis using 10% SDS-PAGE and transferred onto an Amersham Protran Premium 0.45 μm nitrocellulose blotting membrane (GE Healthcare Life Sciences). The membrane was blocked for 2 h at room temperature with 5% nonfat dry milk in TBS-T (25 mM Tris-HCl at pH 7.4, 137 mM NaCl, 2.65 mM KCl, 0.05% Tween-20) and then incubated for 24 h at 4°C with primary antibodies (1:1,000 dilution). After washing with TBS-T, the membrane was incubated for 2 h at room temperature with secondary antibody (1:5,000 dilution). The bands were visualized using a CAS-400SM Davinch-Chemi image™ system (Davinch-K Co. Ltd.).

HT1080 cells were seeded onto a sterilized 24x24 mm, thickness No. 1 Deckglaser microscope cover glass (Paul Marienfel GmbH & Co. KG). The cover glass was then transferred into a 5 cm cell culture dish, and the cells were treated for 30 min at 37°C with I6CA in the presence or absence of PMA (10 ng/ml). For immunofluorescence staining, the cells were washed three times with PBS and fixed for 20 min at room temperature with cold 100% methanol. After removing the methanol and washing three times with PBS, the fixed cells were permeabilized for 20 min at room temperature with 0.5% Tween-20 in PBS. Subsequently, the permeabilized cells were incubated overnight at 4°C with a mouse anti-NF-κB (p65) primary antibody (1:200 diluted in TBS-T). Cells were washed three times with TBS-T and incubated for 2 h at 4°C with an Alexa Fluor 546-conjugated goat polyclonal anti-mouse IgG (H + L) cross-absorbed secondary antibody. Finally, the nuclei were counterstained with Hoechst 33342 (20 ng/ml in PBS) for 5 min at room temperature. The cells were observed under an LSM 700 confocal microscope (Carl Zeiss AG) using a x40 water immersion objective lens. Images were acquired and processed using the ProgRes CapturePro 2.10.0.1 software (Carl Zeiss AG). Pseudo-colors were applied to the images using ImageJ 1.8.0 software, and NF-κB p65 and nuclei were depicted in red and blue, respectively.

All quantitative data are presented in as the mean ± standard deviation of at least three independent experiments that were conducted using fresh reagents. The statistical significance of the differences observed between groups was assessed by analysis of variance, followed by Duncan's multiple range test. All statistical analyses were performed using the SPSS Statistics 12.0 software (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

We first assessed the cytotoxicity of I6CA purified from S. thunbergii extract using an MTT assay. HT1080 cells were treated for 24 h with increasing concentrations of I6CA in the presence or absence of PMA and serum. Following the MTT assay, we found no evidence of a cytotoxic effect of I6CA and PMA on HT1080 cells, even at the highest concentration of 400 μM (Fig. 2). Therefore, it was concluded that these concentrations had no impact on cell viability and could be used to conduct subsequent experiments.

Next, we examined the inhibitory effects of I6CA on the secretion and protein expression of MMP-9 in HT1080 cells. Using gelatin zymography, we assessed the gelatinolytic activity of MMP-9 in the cell culture supernatant of PMA-stimulated HT1080 cells. Notably, we reported that I6CA inhibited the gelatinolytic activity of MMP-9 in a dose-dependent manner (Fig. 3A). To determine whether I6CA inhibited MMP-9 expression, we performed western blot analysis. Our data demonstrated that the expression levels of MMP-9 exhibited a similar trend to the gelatinolytic activity in the cell culture supernatant (Fig. 3B).

To investigate the mechanism mediating the inhibition of MMP-9 expression by I6CA, we used western blotting to analyze whether I6CA could regulate the activation of MAPKs in PMA-stimulated HT1080 cells. MAPK activation is mediated by phosphorylation (8). As shown in Fig. 4A, the phosphorylation of the three MAPKs, JNK, ERK and p38 MAPK, was significantly promoted in PMA-stimulated HT1080 cells, compared with in untreated cells. Of note, I6CA treatment significantly suppressed the phosphorylation of JNK and ERK, but not that of p38 MAPK, in response to PMA stimulation.

We examined whether I6CA decreases nuclear translocation of the NF-κB p65 subunit. As presented in Fig. 4B, western blotting was conducted to analyze the phosphorylation and degradation of IκBα, an essential step in the nuclear translocation of NF-κB p65 subunit (10). We determined that PMA stimulation induced the phosphorylation and degradation of IκBα, and NF-κB nuclear translocation in HT1080 cells, whereas I6CA treatment suppressed these effects induced by PMA. To confirm these results, we directly monitored the nuclear translocation of the NF-κB p65 subunit using immunofluorescence staining and confocal microscopy. When compared with untreated cells, the amount of NF-κB p65 detected in the nuclei of HT1080 cells increased following PMA stimulation (Fig. 5). Conversely, I6CA inhibited the nuclear translocation of NF-κB, as indicated by a reduced level of NF-κB p65 detected in the nuclei of HT1080 cells treated with both I6CA and PMA.