Food Chemistry 141 (2013) 503–509
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Matrix metalloproteinases (MMPs) inhibitory effects of an octameric oligopeptide isolated from abalone Haliotis discus hannai Van-Tinh Nguyen a, Zhong-Ji Qian a, BoMi Ryu b, Kil-Nam Kim c, Daekyung Kim c, Young-Mog Kim d, You-Jin Jeon e, Won Sun Park f, Il-Whan Choi g, Geun Hyung Kim h, Jae-Young Je i,⇑, Won-Kyo Jung a,⇑ a
Department of Marine Life Science and Marine Life Research & Education Center, Chosun University, Gwangju 501-759, Republic of Korea School of Pharmacy, The University of Queensland, Brisbane, Qld 4072, Australia Marine Bio Research Team, Korea Basic Science Institute (KBSI), Jeju 690-140, Republic of Korea d Department of Food Science and Technology, Pukyong National University, Busan 608-737, Republic of Korea e Department of Marine Life Science, Jeju National University, Jeju 690-756, Republic of Korea f Department of Physiology, School of Medicine, Kangwon National University, Chuncheon 200-701, Republic of Korea g Department of Microbiology, College of, Inje University, Busan 614-735, Republic of Korea h Department of Bio-Mechatronic Eng., College of Biotechnology and Bioengineering, Sungkyunkwan UniversitySuwon, Republic of Korea i Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu 550-749, Republic of Korea b c
a r t i c l e
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Article history: Received 26 November 2012 Received in revised form 23 January 2013 Accepted 11 March 2013 Available online 18 March 2013 Keywords: Matrix metalloproteinases (MMP-2/-9) Human fibrosarcoma cells (HT1080) Abalone Haliotis discus hannai Purified abalone oligopeptide (AOP) Nuclear factor-kappaB (NF-jB)
a b s t r a c t Abalone (Haliotis discus hannai) is a marine gastropod, and an important fishery and food industrial resource that is massively maricultured in Asia, Africa, Australia and America. However, its health benefits have rarely been studied for nutraceutical and pharmaceutical application. In this study, the purified abalone oligopeptide (AOP) with anti-matrix metalloproteinases (anti-MMPs) effects was isolated from the digests of abalone intestine using recycle HPLC with a JAI W253 column and an OHpak SB-803 HQ column. The AOP was identified as Ala-Glu-Leu-Pro-Ser-Leu-Pro-Gly (MW = 782.4 Da) with a de novo peptide sequencing technique using a tandem mass spectrometer. The AOP exhibited a specific inhibitory effect against MMP-2/-9 activity and attenuated protein expression of p50 and p65 in the human fibrosarcoma (HT1080) cells, dose-dependently. The results presented illustrate that the AOP could inhibit MMP-2/-9 expression in HT1080 cells via the nuclear factor-kappaB (NF-jB)-mediated pathway. This data suggest that the AOP from H. discus hannai intestine may possess therapeutic and preventive potential for the treatment of MMPs-related disorders such as angiogenesis and cardiovascular diseases. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that play important roles in the degradation of the extracellular matrix, in a variety of biological and pathological processes, as in the invasion and metastasis of tumors (Hwang et al., 2010; Pollitt, Pass, & Pollitt, 1998; Rajapakse, Mendis, Kim, & Kim, 2007). Especially, gelatinase (MMP-2/-9) are known to be involved in processes such as tumour invasion and metastasis (Sang et al., 2006; Schmalfeldt et al., 2001). MMP-2 (gelatinase-A) has been implicated broadly in the invasion and metastasis of many cancer model systems, such as human breast cancer (Jezierska & Tomasz, 2009). MMP-9 (gelatinase B) is thought to play a major role in tumor growth and metastasis, since it has a unique ability to degrade type IV collagen (Jiang & Muschel, 2002; Khan, Kong, ⇑ Corresponding authors. Tel.: +82 61 659 7416, fax: +82 61 659 7419 (J.-Y. Je), tel./fax: +82 62 230 6657 (W.-K. Jung). E-mail addresses:
[email protected] (J.-Y. Je),
[email protected] (W.-K. Jung). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.038
Kim, & Kim, 2010). Moreover, studies have suggested that nuclear factor-kappaB (NF-jB), a key transcription factor for the production of MMP-2/-9, can be activated by various pro-inflammatory cytokines and promotes inflammation (Chou et al., 2010; Park et al., 2007). HT1080 cells have been used extensively in the study of the extracellular matrix proteins involved in attachment, invasion and metastasis. Shellfish, such as mussels, clams and abalones, are a commercially important bioresource in the fishery and food industry. Abalone is a marine gastropod and an important shellfish and industrial resource in Asia, Africa, Australia, and America, and approximately 100 species of abalone are found worldwide (Hintsa, Paul, & Xiao, 2012; Jung, Kim, & Kim, 2007; Qian et al., 2012; Won, Kawamura, Takami, Hoshikawa, & Watanabe, 2011). Pacific abalone (Haliotis discus hannai) has been widely maricultured in East Asia. To meet the increasing demand of the big Asian markets, such as China, Taiwan, Hong Kong and Japan, the total quantity of H. discus hannai production has increased in recent years, mainly due to its high commercial value (Roberto, Alfonso, Andres, & Ricardo, 2007). H. discus hannai abalone mariculture has been
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expanding in land- and sea-based systems and the total yield from South Korea was estimated at 7580 metric tons in 2009. Korea is one of the major suppliers of abalone, and the majority of Korean production is in the remote Wando Island (Cook & Gordon, 2010). In addition, various types of manufacturing products of abalone (dried, steamed, seasoning, spiced abalone, and so on) have also been significantly increased. A great need exists to study the structural, compositional and sequential properties of bioactive peptides. Some recent studies have reported the in vitro formation of bioactive peptides from marine food sources. They have shown the different kinds of bioactivities, such as antibacterial (Schnapp, Kemp, & Smith, 1996), antihypertensive (Je, Park, & Kim, 2005; Jung et al., 2006; Suetsuna, Maekawa, & Chen, 2004) antioxidative, (Jung, Rajapakse, & Kim, 2005) and immunomodulatory effects (Tsuruki et al., 2003). These reports have confirmed that bioactive peptides released by enzymatic proteolysis of food proteins may act as potential physiological modulators of metabolism during intestinal digestion. Bioactive peptides usually contain 3–20 amino acid residues, and their activities are based on their amino acid composition and sequence (Qian, Jung, Byun, & Kim, 2008). Recently, shellfish proteins have been investigated predominantly for the mining of novel peptides with specific or multi-functional bioactivity, such as antimicrobial, anticancer, antioxidant and angiotensin-converting enzyme (ACE) inhibitory effects (Aneiros & Anoland, 2004; Pádraigín & Richard, 2012; Tsai, Chen, & Pan, 2008). As a precious marine product, the nutritional and pharmacological values of abalone have received extensive attention. Abalone has been a valuable food source for humans and the various larger species of abalones have been exploited commercially for food. However, its health benefits have rarely been studied for nutraceutical and pharmaceutical application. Digestion by gastrointestinal proteases can be used as a production process for bioactive peptides, with the advantage that the formed peptides will resist physiological digestion after oral intake. To evaluate the health benefits of abalone, and to utilise abalone, intestines as byproducts discarded from manufacturing processes, we prepared in vitro gastrointestinal digests of abalone intestine. Bioabsorbable and bioactive peptides could be released and modified from the food matrix by the gastrointestinal digestion using digestive enzymes such as pepsin, trypsin, and lipase. MMP-2/-9 inhibitory effects of in vitro gastrointestinal digests from H. discus hannai intestine in HT1080 cells was also evaluated. The scope of this paper is to further investigate the conditions of in vitro gastrointestinal digestion, leading to the formation and/or degradation of bioactive peptides and to elucidate MMPs inhibitory effects. In the present study, we identified its MMP-2/-9 inhibitory effects in HT1080 cells, and purified the bioactive peptides. The accurate molecular mass and amino acid sequence of purified abalone oligopeptide (AOP) was identified by the use of a quadrupole time-of-flight (Q-TOF) mass spectrometer. Furthermore, the AOP has evaluated for its inhibitory effect against expression of MMP2/-9 in HT1080 cells. 2. Materials and methods 2.1. Materials H. discus hannai were collected from Wando Island, South Korea. Intestinal organs (guts) were separated from the washed abalone and lyophilized. Dulbecco’s modified eagle’s medium (DMEM), trypsin–ethylenediaminetetraacetic acid (trypsin–EDTA), penicillin/streptomycin, and foetal bovine serum (FBS) were obtained from Gibco BRL, Life Technologies (Grand Island, NY, USA). HT1080 cells were obtained from American Type of Culture Collec-
tion (Manassas, VA, USA). JAIGEL W253 (20 500 mm) column was purchased from JAI Co., Ltd (Tokyo, Japan). OHpak SB-803 HQ (8 300 mm) column was purchased from Showa Denko K.K. (Tokyo, Japan). Primary and secondary antibodies used for Western blot analysis were MMP-2 (sc-13595), MMP-9 (sc-10737), NF-jB p65 (sc-8008), NF-jB p50 (sc-166588), b-Actin (sc-130656), goat anti-rabbit IgG-HRP (sc-2004) and goat anti-mouse IgG1-HRP (sc2060), and purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Gelatin (type A) and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Other chemicals and reagents used were of analytical grade. 2.2. Preparation of abalone intestine gastrointestinal digests using UF membrane bioreactor systems Preparation of the abalone intestine gastrointestinal digests (AIGID) was carried out following the method described by Kapsokefalou and Miller (1991). Abalone intestine solution was brought to pH 2.2 in gastric digestion using 1 M HCl and 10 M NaOH. Pepsin (EC 3.4.23.1) was added at an enzyme to substrate ratio of 1/100 (w/w), then incubated at 37 °C on a shaker for 2 h. The pH was set to 6.5 to obtain the conditions of small intestinal digestion. Trypsin (EC 3.4.21.4) and a-chymotrypsin (EC 3.4.21.1) were both supplemented at an enzyme to substrate ratio of 1/ 100 (w/w), then incubated at 37 °C for 2.5 h. AIGID was centrifuged at 10,000 g for 15 min at 4 °C and the supernatant was lyophilized to obtain powders (5.65 g). The resultant AIGID was fractionated through two different ultrafiltration (UF) membranes having a range of molecular weight cutoffs (MWCOs) of 100 and 10 kDa as follows: 100 kDa < AIGID I; 10 kDa < AIGID II < 100 kDa; AIGID III < 10 kDa. Finally AIGID I–III were lyophilized. 2.3. Purification of abalone oligopeptide from AIGID III < 10 kDa 2.3.1. Re-cycle chromatography AIGID III was using a JAI W253 (20–500 mm, JAI Co., Ltd, Tokyo, Japan) column. The lyophilized AIGID III (30 mg/ml) was loaded onto the column equilibrated with 0.02 M sodium phosphate buffer (pH 6.8) at a flow rate of 3 ml/min. The eluted peaks were detected at 215 nm, and the active peak was concentrated using a freeze-drier. Active peaks were determined by a gelatin zymography method. 2.3.2. High-performance liquid chromatography (HPLC) The fraction exhibiting the highest inhibitory effect on gelatinase activity was further purified using reversed-phase high-performance liquid chromatography (RP-HPLC) on a Shodex OHpack SB 803 HQ column with a buffer containing 0.01% trifluoroacetic acid (TFA) at flow rate of 0.7 ml/min. The complete course was monitored using a UV detector at 215 nm. Each peak was collected and lyophilized. After the lyophilzation, the samples were then analysed for their gelatinase inhibitory effects. The pure peptide was subjected to amino acid sequence analysis. 2.3.3. Amino acid sequences of purified abalone oligopeptide (AOP) The accurate molecular mass and amino acid sequence of the AOP were determined by the use of a Q-TOF mass spectrometer (Micromass, Altrincham, UK) coupled to an electrospray ionization (ESI) source. The AOP was injected into the electrospray source following dissolution in methanol/water (1:1, v/v), and its molecular mass was determined by a doubly charged (M+2H)2+ state in the mass spectrum. Following molecular mass determination, the peptide was automatically selected for fragmentation and its sequence information was obtained by tandem mass spectroscopy (MS) analysis.
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2.4. Culture of cells and viability determination HT1080 cells were cultured as monolayers in 10 cm culture dishes at 5% CO2 and 37 °C humidified atmosphere. DMEM containing 10% FBS and antibiotics was used as the culture medium for HT1080 cells. The medium was changed 2–3 times each week. To determine cytocompatible effects of the AOP, HT1080 cells were seeded into 96-well plates at a density of about 1 104 cells/well and incubated with different concentrations of samples for 36 h in the presence of serum. After incubation, 50 ll of 1 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent was added to each well and incubation was continued for another 4 h. Mitochondrial succinate dehydrogenase in live cells converts MTT into visible formazan crystals during incubation at 37 °C. The formazan crystals were then solubilised in dimethyl sulfoxide (DMSO) and the optical density was measured at 540 nm by using a microplate reader. Relative cell viability was calculated compared to the non-treated blank group. The data were expressed as means of at least three independent experiments and P < 0.05 was considered significant. 2.5. Determination of MMP-2/-9 activity by gelatin zymography HT1080 cells were cultured on 24-well plates in serum-free DMEM medium. HT1080 cells treated with samples after 1 h stimulation by 10 ng/ml phorbol 12-myristate 13-acetate (PMA). After 36 h, the conditioned medium was collected and centrifuged at 3000 rpm for 10 min to remove cell debris. The gelatinase activities of MMP-2/-9 were determined by gelatin zymography. Concentrated medium was electrophoresed under nonreducing conditions and without heating through 10% sodium dodecyl sulphate (SDS)– polyacrylamide gels impregnated with 0.15% of gelatin. After electrophoresis, SDS is removed from the gel by washing in 2.5% Triton X-100 solution for 1.5 h and incubated in developed buffer [50 mM Tris–HCl buffer (pH 7.5), 200 mM NaCl, 5 mM CaCl22H2O, 0.02% Brif-35] at 37 °C for 36 h. The gels were stained with 1% Coomassie blue R-250 in 45% methanol and 10% glacial acetic acid. After 30 min, the gels were destained in the same solution without the Coomassie blue dye. Proteolytic activity was detected as clear bands against the background stain of undigested substrate in the location of gelatinase.
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unpaired Student’s t-test. The differences were considered statistically significant at P < 0.05. 3. Results 3.1. Preparation of abalone intestine gastrointestinal digests using ultrafiltration membrane bioreactor systems AIGID was fractionated using an UF membrane bioreactor system with range of MWCOs. The AIGIDs were named as AIGID I, which did not pass through the 100 kDa membrane; AIGID II, which passed through the 100 kDa membrane, but not through the 10 kDa; AIGID III, which passed through the 10 kDa membrane. Briefly, three kinds of AIGIDs were designed as follows: 100 kDa < AIGID I; 10 kDa < AIGID II < 100 kDa; AIGID III < 10 kDa. One of them, AIGID III exhibited the most inhibition of MMP-2/-9 activity (Fig. 1A). Thus, AIGID III was selected for next experiments. 3.2. Purification of the AOP and effect on expressions of MMPs In the present study, AIGID III (
