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Introduction

The degradation of the extracellular matrix (ECM) serves an important in various diseases; for instance, in cancer, ECM degradation contributes to tumor invasion or metastasis (1). The matrix metalloproteinases (MMPs) are important members of the zinc-dependent endopeptidase family and have been classified into eight subgroups, which include collagenases, matrilysin, metalloelastase, gelatinases, enamelysin and stromelysins (2). These enzymes target several components of the ECM, such as collagen, elastin and fibronectin; thus, they serve a crucial role in its degradation (3). In addition, MMPs are involved in a variety of physiological and pathological processes, including angiogenesis, tissue remodeling, and tumor invasion and metastasis (4-6). Among the MMPs, MMP-2 and MMP-9 are gelatinases, and have been reported to serve an important role in tumor invasion and metastasis through their ability to degrade an essential ECM component, type IV collagen (7). Notably, previous reports have established that the increased MMP-9 expression in cancer cells is associated with a marked increase in tumor invasion and metastasis, which is mediated by ECM degradation (8-10). Therefore, the targeted inhibition of MMP-9 expression in cancer cells presents a promising approach to suppress ECM degradation, and restrain tumor invasion and metastasis (1,11,12).

Brown algae constitute a rich source of bioactive compounds, which have recently attracted increasing attention due to their biomedical and pharmaceutical potential (13,14). As of the health-beneficial effects of these bioactive compounds, brown algae have been used worldwide, including Korea, Japan, China, and European countries, as a functional food, medicinal ingredients and gelling agents (15,16). Among brown algae, Sargassum thunbergii is one of the main species in biomass, as well as an important pharmaceutical resource (17,18). Accordingly, extracts and bioactive compounds, including polysaccharides, phlorotannins, flavonoids and proteins, which are derived from S. thunbergii have been extensively investigated (18). Furthermore, numerous studies demonstrated that these extracts and compounds exhibit a broad spectrum of biological activities, including pro-osteoblastogenic, antitumor, anti-inflammatory, antioxidative, anti-MMP and antiadipogenic effects (19-28).

Indole derivatives can be produced through chemical synthesis or can be isolated from several natural resources, including Actinobacteria, algae, cruciferous vegetables, fungal and marine sponges (29-32). They exhibit a variety of biological activities, including antitumor, antioxidative, anti-inflammatory and anticonvulsant properties (33-35). In addition, the indole derivative indole-6-carboxaldehyde (I6CA) has been shown to exert anti-adipogenic effects in 3T3-L1 adipo-cytes (26). Although previous reports have highlighted their biological potential, to the best of our knowledge, no study has reported the MMP inhibitory activity of indole derivatives and their underlying mechanism of action.

Therefore, the present study aimed to utilize phorbol 12-myristate 13-acetate (PMA), a compound that can activate MMP-9, to mimic the conditions in cancer cells, and investigate the inhibitory effects of I6CA isolated from S. thunbergii on MMP-9. Furthermore, we aimed to determine the mechanism underlying the effects of I6CA in HT1080 cells.

Materials and methods

Materials

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.

Extraction and isolation of I6CA from S. thumbergii

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%.

Figure 1 Chemical structure of indole-6-carboxaldehyde.

Cell culture

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.

Cell viability assay

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.

Analysis of MMP-9 gelatinolytic activity by gelatin zymography

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.

Western blot analysis

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.).

Immunofluorescence staining and confocal microscopy

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.

Statistical analysis

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.

Results

I6CA does not affect the viability of HT1080 cells

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.

Figure 2 Effects of I6CA on the cell viability of HT1080 cells. The effects of I6CA on HT1080 cell viability (A) with and (B) without PMA-stimulation. N.S., not significant; I6CA, indole-6-carboxaldehyde; PMA, phorbol 12-my ristate 13-acetate.

I6CA inhibits MMP-9 secretion and expression in HT1080 cells

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).

Figure 3 I6CA inhibits MMP-9 secretion and protein expression in PMA-stimulated HT1080 cells. Effects of I6CA on (A) PMA-induced MMP-9 secretion and (B) MMP-9 protein expression in PMA-stimulated HT1080 cells using gelatin zymography and western blotting, respectively. The ratio of densities of the MMP-9 secretion and protein expression bands were used to determine the gelatinolytic activity and relative expression of the MMP-9 protein, respectively. The data are normalized against the ratio calculated for PMA-stimulated HT1080 cells (arbitrarily set to 1-fold). All data are presented as the mean values ± standard deviation of triplicate experiments. *P<0.05 and **P<0.01 vs. PMA-stimulated HT1080 cells. I6CA, indole-6-carboxaldehyde; MMP-9, matrix metalloproteinase-9; PMA, phorbol 12-myristate 13-acetate.

I6CA suppresses mitogen-activated protein kinases (MAPKs) activation in PMA-stimulated HT1080 cells

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.

Figure 4 Inhibitory effects of I6CA on MMP-9-related signaling pathway. Effects of I6CA on phosphorylation of three members of the MAPK family, (A) JNK, ERK and p38 MAPK, and the levels of activation of two essential players in the NF-κB pathway, (B) IκBα and NF-κB p65. The ratio between the densities of the bands corresponding to the proteins of interest and loading control was used to determine their relative expression. The expression data was normalized against the ratio calculated for PMA-stimulated HT1080 cells (arbitrarily set to 1-fold). All data are presented as the mean values ± standard deviation of triplicate experiments. *P<0.05 vs. PMA-stimulated HT1080 cells. #P<0.05 vs. un-stimulated HT1080 cells. ERK, extracellular signal-regulated kinase; I6CA, indole-6-carboxaldehyde; MMP-9, matrix metalloproteinase-9; IκBα, inhibitor of κBα; JNK, c-Jun N-terminal kinase; p, phosphorylated; PMA, phorbol 12-myristate 13-acetate.

I6CA inhibits IκBα degradation and prevents NF-κB nuclear translocation in PMA-stimulated HT1080 cells

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.

Figure 5 I6CA inhibits NF-κB p65 nuclear translocation. The conditions were comprised no treatment, cells treated with 10 ng/ml PMA for 30 min, and the combined treatment of cells with 400 μM I6CA and 10 ng/ml PMA. Red and blue indicated NF-κB cellular distribution and the position of the nucleus, respectively. Scale bar, 50 μm. I6CA, indole-6-carboxaldehyde; NF-κB, nuclear factor-κB; PMA, phorbol 12-myristate 13-acetate.

Discussion

The incidence of cancer-associated mortality is steadily increasing, and tumor metastasis, the formation of secondary tumors in distant organs, constitutes the leading cause of these deaths (40,41). Tumor metastasis is a fundamental property of malignant cells and occurs via a series of sequential events that, in the long term, involves the migration and invasion of neighboring tissues, tumor cell intravasation and survival in the circulation system (blood and lymph), and extravasation and proliferation to form secondary tumors in distant organs (42). Tumor cells must degrade the ECM to migrate and invade surrounding tissues and organs; the suppression of ECM degradation is a crucial step in the prevention of tumor metastasis (1). It is widely recognized that MMP-2 and MMP-9 can degrade various components of the ECM and, therefore, are vital regulators of ECM degradation (43,44). With increasing interest in exploring the applications of medicinal substances extracted from plants and other organisms, various compounds have been evaluated for their potential effects as inhibitors of MMP-2 and MMP-9 activities (45-47).

Indole derivatives extracted from various natural resources, including marine algae, have attracted increasing attention, and numerous studies have investigated their biological effects (29-32). Among indole derivatives, a recent study has shown that I6CA could inhibit obesity-related adipogenesis (26); however, the analysis of the biological effects of I6CA remains limited. Therefore, we purified I6CA from S. thunbergii for further investigation, focusing on its potential effects as an inhibitor of MMP-9, and the underlying mechanisms. In this report, we presented data indicating that I6CA significantly inhibited MMP-9 secretion and expression in PMA-stimulated HT1080 cells. Comparing the observed inhibitory effect of I6CA on MMP-9 with those reported for other bioactive compounds, we noted that bisphosphonates, carboxylated chitooligosaccharides, various cardiovascular drugs and flavonoids were less potent than I6CA purified from S. thunbergii (1,48-50). However, the methanol extract of the red alga Corallina pilulifera appeared to be a more potent inhibitor of MMP-9 than I6CA (51).

The MAPK and NF-κB pathways are generally recognized for their role in the regulation of various physiological processes, including cell proliferation, apoptosis and invasion (3,7,52). According to previous studies, MAPK and NF-κB pathways are known to promote MMP-9 expression in PMA-stimulated HT1080 cells (42,53). The MAPK pathways involve the phosphorylation and subsequent activation of MAPK family members, including JNK, ERK, and p38 MAPK (54). In the NF-κB pathway, IκBα binds and retains in the cytosol the inactivated form of the canonical p65/p50 heterodimer, which is activated and translocates to the nucleus upon IκBα phosphorylation, ubiquitination and degradation (55-58). In previous studies, PMA has been shown to activate the MAPK and NF-κB pathways in HT1080 cells (7,8,59-61). Therefore, the activation state of the MAPK and NF-κB pathways is often assessed to analyze the mechanism of regulation of MMP-9 expression (8,62). To unravel the mechanisms of MMP-9 inhibition by I6CA, we examined the activation state of the MAPK and NF-κB pathways in PMA-stimulated HT1080 cells. Our results indicated that I6CA suppressed the phosphorylation of JNK and ERK, but not that of p38 MAPK, in response to PMA stimulation. In addition, I6CA inhibited the phosphorylation and degradation of IκBα, and NF-κB p65 nuclear translocation in PMA-stimulated HT1080 cells. Previous studies reported the MMP inhibitory effects of various bioactive compounds, such as sulfated polysaccharides and polyphenolic compounds isolated from brown algae (63-65). Based on our findings, we proposed that I6CA, an indole derivative isolated from a brown alga, is a potent MMP-9 inhibitor that acts via the suppression of the MAPK and NF-κB pathways.

In conclusion, we demonstrated that I6CA purified from S. thunbergii significantly inhibited the secretion and protein expression of MMP-9 in PMA-stimulated HT1080 cells. These effects were determined to be mediated via the suppression of phosphorylation and activation of JNK and ERK, IκBα phosphorylation and degradation, and NF-κB p65 nuclear translocation (Fig. 6). These findings may further our understanding of the mechanism of action of I6CA in the inhibition of MMP-9. Of note, we investigated only the inhibitory effects of I6CA on MMP-9. Therefore, we plan to perform in vivo experiments in the future to verify whether I6CA inhibits ECM degradation through MMP-9 inhibition, suppressing metastasis.

Figure 6 Schematic diagram of the mechanism underlying the inhibitory effects of indole-6-carboxaldehyde on MMP-9 in PMA-stimulated HT1080 cells. ERK, extracellular signal-regulated kinase; IκBα, inhibitor of κBα; JNK, c-Jun N-terminal kinase; MMP-9, matrix metalloproteinase-9; NF-κB, nuclear factor-κB; p, phosphorylated; PMA, phorbol 12-myristate 13-acetate.

Acknowledgements

Not applicable.

Funding

This research was supported by a research grant from the Marine Biotechnology Program (grant. no. 20150220) of the Ministry of Oceans and Fisheries of Republic of Korea and partially supported by a research grant from the Korea Institute of Ocean Science and Technology (grant. no. PE99722).

Availability of data and materials

The datasets used and/or analyzed in the present study are available from the corresponding author on reasonable request.

Authors' contributions

THK, SJH and SCK performed the experiments. SJH extracted and isolated indol-6-carboxaldehyde. MY, WSP, IWC and WKJ conceived and designed the study. All authors have reviewed and approved the final version of the manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References