Inhibition of MMP-2 and MMP-9 activities by solvent-partitioned Sargassum horneri extracts
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Matrix metalloproteinases (MMPs) are linked with several complications such as metastasis of cancer progression, oxidative stress, and hepatic fibrosis. Brown seaweeds are being extensively studied for their bioactive molecule content against cancer progression. In this context, Sargassum horneri was reported to possess various bioactivities including antiviral, antimicrobial, and anti-inflammatory partly due to its phenolic compound content.
In this study, potential of S. horneri was evaluated through anti-MMP effect in HT1080 fibrosarcoma cells. S. horneri crude extract was fractionated with organic solvents, namely, water (H2O), n-buthanol (n-BuOH), 85% aqueous methanol (85% aq. MeOH), and n-hexane. The non-toxicity of fraction samples (Sargassum horneri solvent-partitioned extracts (SHEs)) was confirmed by cell-viability assay. SHEs were tested for their ability to inhibit MMP enzymatic activity through gelatin digestion evaluation and cell migration assay. Expressions of MMP-2 and MMP-9 and tissue inhibitors of MMP (TIMPs) were evaluated by reverse transcription and Western blotting.
All fractions inhibited the enzymatic activities of MMP-2 and MMP-9 according to gelatin zymography. Except H2O fraction, fractions hindered the cell migration significantly. All tested fractions suppressed both mRNA and protein levels of MMP-2, MMP-9, TIMP-1, and TIMP-2.
Overall, current results suggested that S. horneri has potential to be a good source for anti-MMP agents, and further investigations are underway for better understanding of the action mechanism and isolation and elucidation of the bioactive molecules.
KeywordsBrown algae MMP Sargassum horneri TIMP
Phorbol 12-myristate 13-acetate
Sargassum horneri solvent-partitioned extract
Tissue inhibitor of matrix metalloproteinase
Matrix metalloproteinases are hailed as crucial enzymes that have important roles in cancer progression and several tumor-related complications (Jones and Walker 1997). Due to their nature, matrix metalloproteinases (MMPs) are pivotal in cell proliferation and migration which are closely linked with invasive tumor cells and onset of malignant tumor growth (Moss et al. 2012). In addition, MMPs which are zinc-dependent endopeptidases responsible for extracellular matrix degradation are known to be involved heavily in various disorders including inflammatory response, cardiovascular diseases, arthritis, and most types of cancer (Bauvois 2012; Egeblad and Werb 2002; Overall and López-Otín 2002). Different classifications of MMPs occur depending on the tissues they are found, their functions, and their expression patterns. There are several types of extracellular matrix-related enzymes defined in human cellular mechanisms. Among them, MMP-2 (72 kDa) and MMP-9 (92 kDa) are found to regulate tumor invasion and metastasis. Researches in malignant tumors reported the overexpression and enhanced activity of both of these MMPs (Ibañez and Cifuentes 2013). Enhanced metastasis is evidently linked with enhanced MMP-2 and MMP-9 expressions. Therefore, studies on hindering or inhibiting the expression or activity of MMPs gained interest and put several MMP types including MMP-2 and MMP-9 into spotlight as therapeutic targets. MMPs are regulated by tissue inhibitor of MMP (TIMP) through a negative feedback mechanism acted on activation of MMP enzymes. TIMP expression inhibits all types of MMPs other than gelatinases. Normal regulation mechanism of TIMPs ought to be deteriorated and further facilitate the enhancement of MMP expression in malignant tumors (Yu and Gu 2015).
Brown macroalgae have been of much interest due to their known ability to withstand different marine environments by producing various secondary metabolites. Some brown algae species already have been reported to possess numerous health beneficial effects (Holdt and Kraan 2011; Matanjun et al. 2009; Patarra et al. 2011). Low content in harmful lipids and high content in polysaccharides, unsaturated fatty acids, vitamins, and minerals enable marine algae to be promoted as a nutritious source for healthy diets (Plaza et al. 2010). Sargassum horneri is a common species of brown macroalgae that grows on the coastal sea of Korea and Japan. Although it has been a part of a diet in limited areas of Japan, it could not enter the market as a processed product until recently. Literature contains little information about its chemical composition and nutritional aspects (de la Mare et al. 2012; Thomas and Kim 2011). Extracts from S. horneri were observed to stimulate the formation of bone tissue and to prevent bone loss (Jiao et al. 2009). In addition, chromene isolated from S. horneri was reported to possess a protective effect against UV-A-induced damage in skin dermal fibroblasts (Reuter et al. 2010). In this context, as a part of ongoing research to develop antitumor compounds, especially MMP inhibitors from natural origin, current study aims to present understanding on the potential of S. horneri as a source for natural products that can act on MMP activity. In this manner, solvent-partitioned fractions of S. horneri extract were screened for their effects on MMP-2 and MMP-9 activity and expression.
Plant materials and fractionation
S. horneri was purchased from Parajeju (Jeju, Korea) in 2013. The sample (1000 g) was air-dried outdoors under shade, ground to powder using a blender (NFM-3561SN, NUC Co. Ltd., Seoul, Korea) at high-speed grinding setting, and extracted in a 5-l Erlenmeyer flask with EtOH (3 l) for three times. The collected extracts were concentrated under reduced pressure with a rotary evaporator (80 mbar, 50 °C) (RV 10 Series, IKA, Wilmington, NC, USA).
The crude extract (128 g) was suspended in between CH2Cl2 and water. Later, the CH2Cl2 layer was separated by 85% aqueous MeOH and n-hexane. Next, the water layer was partitioned with n-BuOH and H2O, respectively. Overall, the solvent partition yielded the n-hexane (0.73 g), 85% aq. MeOH (4.05 g), n-BuOH (1.08 g), and H2O (71.32 g) fractions.
Cell culture and cell viability determination
HT1080 human fibrosarcoma cells were cultured in T-75 culture flasks (Nunc, Roskilde, Denmark) in an incubator with 37 °C and 5% CO2 atmosphere with Dulbecco’s modified Eagle’s medium (DMEM; Gibco-BRL, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 μg/ml penicillin-streptomycin (Gibco-BRL, Gaithersburg, MD, USA). The medium was changed every 3 days.
For cell viability assessment, cells were cultured in 96-well plates at a 5 × 103 cells/well density. Following a 24-h incubation, cell culture medium was removed and the cells were washed with fresh medium and prior to treatment with the medium with or without S. horneri samples. After incubation for 48 h, cells were re-washed with fresh medium and 100 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (1 mg/ml) was introduced to the wells, followed by a 4-h incubation. Finally, 100 μl of dimethyl sulfoxide (DMSO) was used for each well in order to solubilize the formazan crystals prior to absorbance measurement at 540 nm using a GENios® microplate reader (Tecan Austria GmbH, Grödig, Austria). Cell viability was defined by the absorbance value as a way to indicate the amount of MTT converted into formazan crystal. Viability of cells was determined as a percentage in comparison to the untreated control wells against sample-treated wells, and dose response curves were established.
Cell migration assay
Cells were grown on a 12-well culture dish to 90% confluence followed by forming an injury line with a width of 2 mm from scraping vertically across the cell layer with a sterile scraper. Floating cell debris was washed away with phosphate-buffered saline (PBS), and cell medium was changed to serum-free medium. Cells were treated with 50 μg/ml Sargassum horneri solvent-partitioned extracts (SHEs). Cell migration was observed under an inverted microscope (Nikon Eclipse TS100, Nikon Instruments, Melville, NY, USA), and photographs were taken at incubation starting time and after 24 h of incubation.
MMP enzymatic activity determination by gelatin zymography
Enzymatic activities of MMP-2 and MMP-9 from HT1080 cells that were treated with or without samples were detected by gelatin zymography. HT1080 cells were cultured in 24-well plates with a density of 2 × 105 cells/well in a serum-free medium and were introduced to different concentrations of sample for 1 h. Phorbol 12-myristate 13-acetate (PMA; 10 ng/ml) was used to enhance the MMP expression, and cells were further incubated for 24 h after PMA treatment. Total protein contents of the cells were normalized using Bradford protein determination method. Next, cell culture medium was subjected to substrate-gel electrophoresis. Conditioned cell culture medium with the same amount of protein was transferred on 10% polyacrylamide gels under non-reducing conditions containing 1.5 mg/ml gelatin. Polyacrylamide gels were then washed with 50 mM Tris–HCl (pH 7.5) containing 2.5% Triton X-100 to remove any remaining sodium dodecyl sulfate. After the washing process, gels were incubated for 48 h at 37 °C in developing buffer (10 mM CaCl2, 50 mM Tris–HCl, 150 mM NaCl) in order to facilitate gelatin digestion. Areas of gelatin hydrolyzation by MMP were observed as clear zones against blue background of Coomassie Blue staining under a CAS-400SM Davinch-Chemi imager™ (Davinch-K, Seoul, Korea).
RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) analysis
Total cellular RNA was extracted by TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) from sample-treated and control wells. Any changes in the concentration of mRNA for MMP-2 and MMP-9 were determined by RT-PCR. Briefly, 2 μg of total RNA from cells was converted to single-stranded cDNA using a reverse transcription system (Promega, Madison, WI, USA). The target cDNA was amplified using the following primers: forward 5′-TGA-AGG-TCG-GTG-TGA-ACG-GA-3′ and reverse 5′-CAT-GTA-GCC-ATG-AGG-TCC-ACC-AC-3′ for MMP-2; forward 5′-CAC-TGT-CCA-CCC-CTC-AGA-GC-3′ and reverse 5′-CAC-TTG-TCG-GCG-ATA-AGG-3′ for MMP-9; forward 5′-AAT-TCC-GAC-CTC-GTC-ATC-AG-3′ and reverse 5′-TGC-AGT-TTT-CCA-GCA-ATG-AG-3′ for TIMP-1; forward 5′-TGA-TCC-ACA-CAC-GTT-GGT-CT-3′ and reverse 5′-TTT-GAG-TTG-CTT-GCA-GGA-TG-3′ for TIMP-2; and forward 5′-GCC-ACC-CAG-AAG-ACT-GTG-GAT-3′ and reverse 5′-TGG-TCC-AGG-GTT-TCT-TAC-TCC-3′ for β-actin. Cycles were 95 °C for 45 s, 60 °C for 1 min, and 72 °C for 45 s for amplification. Following the completion of 30 cycles, the final products were separated by electrophoresis on 1.5% agarose gel for 30 min at 100 V. Gel staining was carried out with 1 mg/ml EtBr, and visualization by UV light using AlphaEase® gel image analysis software was finalized with the assay (Alpha Innotech, San Leandro, CA, USA).
Western blot analysis
Immunoblotting was performed according to common standard procedures. To explain briefly, HT1080 cells were agitated in RIPA lysis buffer (Sigma-Aldrich Corp., St. Louis, USA) at 4 °C for 30 min. Cell lysates (35 μg) were then subjected to separation using 10% SDS-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biosciences., England, UK), blocking with 5% skim milk and hybridization with primary antibodies (diluted 1:1000). Membranes were then incubated with horseradish-peroxidase-conjugated secondary antibodies at room temperature. Immunoreactive proteins were detected using an electrochemiluminescence kit (Amersham Pharmacia Biosciences, England, UK) according to the manufacturer’s instructions. Protein bands were observed using a CAS-400SM Davinch-Chemi imager™ (Davinch-K, Seoul, Korea).
The data were presented as a mean of three different experiments ± SD. Differences between the calculated means of the each individual group were determined by one-way ANOVA coupled with Duncan’s multiple range tests. Any difference was considered statistically significant at p < 0.05. The statistical software SAS v9.1 (SAS Institute Inc., Cary, NC, USA) was used for analyses.
Results and discussion
Particular important pathways for metastasis, oxidative stress, and fibrosis are known to be influenced by the activities of MMPs (Holdt and Kraan 2011; Plaza et al. 2010). Therefore, MMP inhibitors steadily gain high interest from various research and development studies of pharma and nutraceutical approaches. Recently, natural sources for MMP-inhibiting substances are being intensively studied, and in this context, marine organisms hold a great deal of potential being present in a unique and challenging environment. Various organisms, especially marine plants, and metabolites have been identified as potential MMP inhibitors, and possible mechanisms of action for isolated compounds have been suggested (de la Mare et al. 2012; Thomas and Kim 2011). In order to provide valuable insights on that matter, S. horneri was studied to evaluate its MMP-inhibition efficiency. In order to help the future utilization through isolated and elucidated bioactive substances, crude extract of S. horneri was fractioned with organic solvents and solvent-partitioned extracts were tested separately.
Possible chemical composition of the 85% aq. MeOH SHE, the most active sample according to current results, was suggested to be formed mostly with phenol-based compounds which are common bioactive substances of brown algae with health beneficial effects (Bhatnagar and Kim 2010) while H2O and n-BuOH SHE possibly contain more glycol-based compounds. Some MMP-inhibiting polysaccharides (Tu et al. 2008) and benzopyran derivates from different sources were already isolated and reported while similar compounds were also found in S. horneri with different bioactivities. Nevertheless, current results indicated that S. horneri is a source for MMP inhibitors that might lead to future development of anti-tumor compounds. On the other hand, detailed evaluation of S. horneri and its constituents will also provide valuable insights for its utilization as functional food, and future studies on its detail action mechanism are urged for a better understanding of its potential. In the current state, S. horneri was suggested as a potential nutraceutical due to its potential anti-MMP effect.
This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology [grant number NRF-2014R1A1A2059310].
Availability of data and materials
All datasets and materials are available from the corresponding author on a reasonable request.
FK and SGL carried out the experiments. JAK, and CSK conceived the idea and designed the experiments. FK, JHO and JAK participated to write the manuscript and interpreted the data. JHO performed the necessary revisions and updates on the manuscript. All authors read and approved the final manuscript.
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