Food Chemistry 141 (2013) 503–509

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Food Chemistry

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Matrix metalloproteinases (MMPs) inhibitory effects of an octamericoligopeptide isolated from abalone Haliotis discus hannai

0308-8146/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.03.038

⇑ 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: jjy1915@chonnam.ac.kr (J.-Y. Je), wkjung@chosun.ac.kr (W.-K.Jung).

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 Koreab School of Pharmacy, The University of Queensland, Brisbane, Qld 4072, Australiac Marine Bio Research Team, Korea Basic Science Institute (KBSI), Jeju 690-140, Republic of Koread Department of Food Science and Technology, Pukyong National University, Busan 608-737, Republic of Koreae Department of Marine Life Science, Jeju National University, Jeju 690-756, Republic of Koreaf Department of Physiology, School of Medicine, Kangwon National University, Chuncheon 200-701, Republic of Koreag Department of Microbiology, College of, Inje University, Busan 614-735, Republic of Koreah Department of Bio-Mechatronic Eng., College of Biotechnology and Bioengineering, Sungkyunkwan UniversitySuwon, Republic of Koreai Department of Marine Bio-Food Sciences, Chonnam National University, Yeosu 550-749, Republic of Korea

a r t i c l e i n f o

Article history:Received 26 November 2012Received in revised form 23 January 2013Accepted 11 March 2013Available online 18 March 2013

Keywords:Matrix metalloproteinases (MMP-2/-9)Human fibrosarcoma cells (HT1080)Abalone Haliotis discus hannaiPurified 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 industrialresource that is massively maricultured in Asia, Africa, Australia and America. However, its health bene-fits have rarely been studied for nutraceutical and pharmaceutical application. In this study, the purifiedabalone oligopeptide (AOP) with anti-matrix metalloproteinases (anti-MMPs) effects was isolated fromthe digests of abalone intestine using recycle HPLC with a JAI W253 column and an OHpak SB-803 HQcolumn. The AOP was identified as Ala-Glu-Leu-Pro-Ser-Leu-Pro-Gly (MW = 782.4 Da) with a de novopeptide sequencing technique using a tandem mass spectrometer. The AOP exhibited a specific inhibitoryeffect against MMP-2/-9 activity and attenuated protein expression of p50 and p65 in the human fibro-sarcoma (HT1080) cells, dose-dependently. The results presented illustrate that the AOP could inhibitMMP-2/-9 expression in HT1080 cells via the nuclear factor-kappaB (NF-jB)-mediated pathway. Thisdata suggest that the AOP from H. discus hannai intestine may possess therapeutic and preventive poten-tial for the treatment of MMPs-related disorders such as angiogenesis and cardiovascular diseases.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Matrix metalloproteinases (MMPs) are a family of zinc-depen-dent endopeptidases that play important roles in the degradationof the extracellular matrix, in a variety of biological and patholog-ical processes, as in the invasion and metastasis of tumors (Hwanget al., 2010; Pollitt, Pass, & Pollitt, 1998; Rajapakse, Mendis, Kim, &Kim, 2007). Especially, gelatinase (MMP-2/-9) are known to be in-volved in processes such as tumour invasion and metastasis (Sanget al., 2006; Schmalfeldt et al., 2001). MMP-2 (gelatinase-A) hasbeen implicated broadly in the invasion and metastasis of manycancer model systems, such as human breast cancer (Jezierska &Tomasz, 2009). MMP-9 (gelatinase B) is thought to play a majorrole in tumor growth and metastasis, since it has a unique abilityto degrade type IV collagen (Jiang & Muschel, 2002; Khan, Kong,

Kim, & Kim, 2010). Moreover, studies have suggested that nuclearfactor-kappaB (NF-jB), a key transcription factor for the produc-tion of MMP-2/-9, can be activated by various pro-inflammatorycytokines and promotes inflammation (Chou et al., 2010; Parket al., 2007). HT1080 cells have been used extensively in the studyof the extracellular matrix proteins involved in attachment, inva-sion and metastasis.

Shellfish, such as mussels, clams and abalones, are a commer-cially important bioresource in the fishery and food industry. Aba-lone is a marine gastropod and an important shellfish andindustrial resource in Asia, Africa, Australia, and America, andapproximately 100 species of abalone are found worldwide (Hint-sa, Paul, & Xiao, 2012; Jung, Kim, & Kim, 2007; Qian et al., 2012;Won, Kawamura, Takami, Hoshikawa, & Watanabe, 2011). Pacificabalone (Haliotis discus hannai) has been widely maricultured inEast Asia. To meet the increasing demand of the big Asian markets,such as China, Taiwan, Hong Kong and Japan, the total quantity ofH. discus hannai production has increased in recent years, mainlydue 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 fromSouth Korea was estimated at 7580 metric tons in 2009. Korea isone of the major suppliers of abalone, and the majority of Koreanproduction is in the remote Wando Island (Cook & Gordon,2010). In addition, various types of manufacturing products of aba-lone (dried, steamed, seasoning, spiced abalone, and so on) havealso been significantly increased.

A great need exists to study the structural, compositional andsequential properties of bioactive peptides. Some recent studieshave reported the in vitro formation of bioactive peptides frommarine food sources. They have shown the different kinds of bioac-tivities, such as antibacterial (Schnapp, Kemp, & Smith, 1996), anti-hypertensive (Je, Park, & Kim, 2005; Jung et al., 2006; Suetsuna,Maekawa, & Chen, 2004) antioxidative, (Jung, Rajapakse, & Kim,2005) and immunomodulatory effects (Tsuruki et al., 2003). Thesereports have confirmed that bioactive peptides released by enzy-matic proteolysis of food proteins may act as potential physiolog-ical modulators of metabolism during intestinal digestion.Bioactive peptides usually contain 3–20 amino acid residues, andtheir activities are based on their amino acid composition and se-quence (Qian, Jung, Byun, & Kim, 2008). Recently, shellfish proteinshave been investigated predominantly for the mining of novel pep-tides with specific or multi-functional bioactivity, such as antimi-crobial, anticancer, antioxidant and angiotensin-convertingenzyme (ACE) inhibitory effects (Aneiros & Anoland, 2004;Pádraigín & Richard, 2012; Tsai, Chen, & Pan, 2008). As a preciousmarine product, the nutritional and pharmacological values of aba-lone have received extensive attention. Abalone has been a valu-able food source for humans and the various larger species ofabalones have been exploited commercially for food. However,its health benefits have rarely been studied for nutraceutical andpharmaceutical application.

Digestion by gastrointestinal proteases can be used as a produc-tion process for bioactive peptides, with the advantage that theformed peptides will resist physiological digestion after oral in-take. To evaluate the health benefits of abalone, and to utilise aba-lone, intestines as byproducts discarded from manufacturingprocesses, we prepared in vitro gastrointestinal digests of abaloneintestine. Bioabsorbable and bioactive peptides could be releasedand modified from the food matrix by the gastrointestinal diges-tion using digestive enzymes such as pepsin, trypsin, and lipase.MMP-2/-9 inhibitory effects of in vitro gastrointestinal digests fromH. discus hannai intestine in HT1080 cells was also evaluated. Thescope of this paper is to further investigate the conditions ofin vitro gastrointestinal digestion, leading to the formation and/ordegradation of bioactive peptides and to elucidate MMPs inhibi-tory effects.

In the present study, we identified its MMP-2/-9 inhibitory ef-fects in HT1080 cells, and purified the bioactive peptides. The accu-rate molecular mass and amino acid sequence of purified abaloneoligopeptide (AOP) was identified by the use of a quadrupoletime-of-flight (Q-TOF) mass spectrometer. Furthermore, the AOPhas evaluated for its inhibitory effect against expression of MMP-2/-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 abaloneand lyophilized. Dulbecco’s modified eagle’s medium (DMEM),trypsin–ethylenediaminetetraacetic acid (trypsin–EDTA), penicil-lin/streptomycin, and foetal bovine serum (FBS) were obtainedfrom 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) columnwas purchased from JAI Co., Ltd (Tokyo, Japan). OHpak SB-803HQ (8 � 300 mm) column was purchased from Showa Denko K.K.(Tokyo, Japan). Primary and secondary antibodies used for Westernblot analysis were MMP-2 (sc-13595), MMP-9 (sc-10737), NF-jBp65 (sc-8008), NF-jB p50 (sc-166588), b-Actin (sc-130656), goatanti-rabbit IgG-HRP (sc-2004) and goat anti-mouse IgG1-HRP (sc-2060), and purchased from Santa Cruz Biotechnology Inc. (SantaCruz, CA, USA). Gelatin (type A) and phorbol 12-myristate 13-ace-tate (PMA) were purchased from Sigma Chemical Co. (St. Louis,MO, USA). Other chemicals and reagents used were of analyticalgrade.

2.2. Preparation of abalone intestine gastrointestinal digests using UFmembrane bioreactor systems

Preparation of the abalone intestine gastrointestinal digests (AI-GID) was carried out following the method described by Kap-sokefalou and Miller (1991). Abalone intestine solution wasbrought to pH 2.2 in gastric digestion using 1 M HCl and 10 MNaOH. Pepsin (EC 3.4.23.1) was added at an enzyme to substrateratio 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 intestinaldigestion. 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 centrifugedat 10,000 � g for 15 min at 4 �C and the supernatant was lyophi-lized to obtain powders (5.65 g). The resultant AIGID was fraction-ated through two different ultrafiltration (UF) membranes having arange of molecular weight cutoffs (MWCOs) of 100 and 10 kDa asfollows: 100 kDa < AIGID I; 10 kDa < AIGID II < 100 kDa; AIGIDIII < 10 kDa. Finally AIGID I–III were lyophilized.

2.3. Purification of abalone oligopeptide from AIGID III < 10 kDa

2.3.1. Re-cycle chromatographyAIGID III was using a JAI W253 (20–500 mm, JAI Co., Ltd, Tokyo,

Japan) column. The lyophilized AIGID III (30 mg/ml) was loadedonto the column equilibrated with 0.02 M sodium phosphate buf-fer (pH 6.8) at a flow rate of 3 ml/min. The eluted peaks were de-tected at 215 nm, and the active peak was concentrated using afreeze-drier. Active peaks were determined by a gelatin zymogra-phy method.

2.3.2. High-performance liquid chromatography (HPLC)The fraction exhibiting the highest inhibitory effect on gelatin-

ase activity was further purified using reversed-phase high-perfor-mance liquid chromatography (RP-HPLC) on a Shodex OHpack SB803 HQ column with a buffer containing 0.01% trifluoroacetic acid(TFA) at flow rate of 0.7 ml/min. The complete course was moni-tored using a UV detector at 215 nm. Each peak was collectedand lyophilized. After the lyophilzation, the samples were thenanalysed for their gelatinase inhibitory effects. The pure peptidewas 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 fol-lowing dissolution in methanol/water (1:1, v/v), and its molecularmass was determined by a doubly charged (M+2H)2+ state in themass spectrum. Following molecular mass determination, the pep-tide was automatically selected for fragmentation and its sequenceinformation 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 culturedishes at 5% CO2 and 37 �C humidified atmosphere. DMEM con-taining 10% FBS and antibiotics was used as the culture mediumfor HT1080 cells. The medium was changed 2–3 times each week.

To determine cytocompatible effects of the AOP, HT1080 cellswere seeded into 96-well plates at a density of about 1 � 104 -cells/well and incubated with different concentrations of samplesfor 36 h in the presence of serum. After incubation, 50 ll of1 mg/ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) reagent was added to each well and incubationwas continued for another 4 h. Mitochondrial succinate dehydro-genase in live cells converts MTT into visible formazan crystalsduring incubation at 37 �C. The formazan crystals were then solu-bilised in dimethyl sulfoxide (DMSO) and the optical density wasmeasured at 540 nm by using a microplate reader. Relative cell via-bility was calculated compared to the non-treated blank group. Thedata were expressed as means of at least three independent exper-iments 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-freeDMEM medium. HT1080 cells treated with samples after 1 h stim-ulation by 10 ng/ml phorbol 12-myristate 13-acetate (PMA). After36 h, the conditioned medium was collected and centrifuged at3000 rpm for 10 min to remove cell debris. The gelatinase activitiesof MMP-2/-9 were determined by gelatin zymography. Concen-trated medium was electrophoresed under nonreducing conditionsand without heating through 10% sodium dodecyl sulphate (SDS)–polyacrylamide gels impregnated with 0.15% of gelatin. After elec-trophoresis, SDS is removed from the gel by washing in 2.5% TritonX-100 solution for 1.5 h and incubated in developed buffer [50 mMTris–HCl buffer (pH 7.5), 200 mM NaCl, 5 mM CaCl2�2H2O, 0.02%Brif-35] at 37 �C for 36 h. The gels were stained with 1% Coomassieblue R-250 in 45% methanol and 10% glacial acetic acid. After30 min, the gels were destained in the same solution without theCoomassie blue dye. Proteolytic activity was detected as clearbands against the background stain of undigested substrate inthe location of gelatinase.

2.6. Western blot assay

HT1080 cells were plated in a 6-well plate and treated with theAOP. They were then washed with phosphate buffered saline (PBS)and lysed, and protein concentration was determined by thebicinchoninic acid (BCA) method using bovine serum albumin asstandard. Proteins were separated by electrophoresis on 10%SDS–polyacrylamide gels and transferred to a nitrocellulose mem-brane. Membranes were blocked with 5% skim milk in Tris bufferedsaline containing Tween-20 (TBS-T) (20 mM Tris–HCl, pH 7.6,136 mM NaCl, and 0.1% Tween-20) and then incubated with theprimary antibody (1:500 dilution) in blocking agent at 4 �C over-night. After washing with TBS-T buffer, the membrane was incu-bated with a secondary antibody (1:5000 dilution) for 2 h atroom temperature. Bands were visualised by enhanced chemilumi-nescence and LAS-4000 imaging system (FUJIFILM, Tokyo, Japan).The basal levels of MMP-2/-9 protein expression were normalisedto b-actin level.

2.7. Statistical analysis

The results are presented as the mean ± standard deviation. Sig-nificant differences among the groups were determined using the

unpaired Student’s t-test. The differences were considered statisti-cally significant at P < 0.05.

3. Results

3.1. Preparation of abalone intestine gastrointestinal digests usingultrafiltration membrane bioreactor systems

AIGID was fractionated using an UF membrane bioreactor sys-tem 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 throughthe 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/-9activity (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 (<10 kDa) was fractionated andfractions I–VII (FrI–FrVII) were collected (Fig. 1B). During this pro-cess of identifying the inhibition of MMP-2/-9 activity, a gelatinzymography assay was used. PMA-stimulated conditioned med-ium was treated with FrI–FrVII at a concentration of 200 lg/ml.When these fractions were tested for gelatinase activity, FrV wasfound to possess a strong inhibitory effect on MMP-2/-9 expressionin HT1080, as compared with PMA treatment groups (Fig. 1C).

The active FrV was further separated by RP-HPLC and the sub-fractions called FrV-1 to FrV-5 were collected (Fig. 2A). The activefour peaks were determined by gelatin zymography method.Among FrV treatment groups, the intensity of the gelatinolyticband of FrV-2 treatment groups was reduced significantly com-pared to the PMA treatment group (Fig. 2B and Fig. 2C). In the nextsteps, the peptide purified from fraction of AIGID III was analysedfor its amino acid sequence. FrV-2, the AOP exerted a clear inhibi-tory effect on MMP-2/-9 activity by gelatin zymography. The ami-no acid sequence of the AOP was Ala-Glu-Leu-Pro-Ser-Leu-Pro-Glywith a molecular size of 782.4 Da (Fig. 2D). The structure of theAOP derived from the digests of abalone intestine is shown inFig. 2E, which is characterised by one acidic residue (Glu), and non-polar residues (Ala, Leu and Pro).

3.3. Effect of the AOP on cell viability of HT1080

FBS is the most widely used for cell culture media because of itshigh content of growth promoting factors. However, in our study,the culture medium was replaced with 0.5 ml of fresh serum-freeDMEM in zymography method and 1 ml of fresh DMEM containing10% FBS in Western blot analysis before the treatment with sam-ple. Thus, to examine cytotoxicity effects of the AOP on HT1080cells, an MTT assay was carried out following treatment with dif-ferent concentration for 36 h in the presence or absence of FBS.All concentrations of the AOP did not any cytotoxic effect onHT1080 even at the highest concentration (100 lM) as shown inFig. 3. Briefly, the MTT assay was demonstrated that the AOP didnot inhibit cell proliferation in the presence or absence of FBS after36 h culture. The viability data clearly confirmed that cytotoxicitydid not contribute to the observed MMP-2/-9 inhibitory potentialof AOP.

3.4. Effect of the AOP on MMP-2/-9 protein expression and NF-jBactivity in HT1080 cells

The aim of this research was to examine whether FrV-2 (theAOP) can specifically inhibit MMP-2/-9 expression. As depicted in

Fig. 1. (A) Gelatin zymography for the determination of MMP-2/-9 activities inAIGID I–III treated HT1080 cell line. (B) Separation of the abalone intestinegastrointestinal digests III was using a JAI W253 (20–500 mm) column at a flow rateof 3 ml/min. The eluted peaks were detected at 215 nm and fraction volumecollected. (C) Gelatin zymography for the determination of MMP-2/-9 activities infractions of AIGID III treated HT1080 cell line. (D) Areas and relative intensities ofgelatin-digested bands by MMP-2/-9. After culture medium of HT1080 cells treatedwith 200 lg/ml of FrI–FrVII for 1 h and stimulated by PMA (10 ng/ml). The resultspresented are the mean ± S.D. of a triplicate experiment. P < 0.05; P < 0.01.

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Fig. 4, a clear concentration dependent suppression effect of theAOP (10, 50 and 100 lM) was observed in MMP-2/-9 protein levelscompared to PMA treatments (control). The inhibition levels ofMMP expressions were more noticeable for MMP-2/-9 treatmentwith AOP at 100 lM. However, the protein expression level ofMMP-2 was higher than that of MMP-9 in HT1080 cells.

Further, to better understand whether the AOP could attenuateprotein expression of p50 and p65, two subunits of NF-jB tran-scription, p50 and p65 protein levels in cytosol were studied using

Western blot analysis. The purified AOP is a low-molecular-weightpeptide (782.4 Da), which could attenuate protein expression ofp50 and p65 in a dose-dependent manner (10, 50 and 100 lM)(Fig. 5). In addition, the AOP exhibited a higher inhibitory effecton p50 activity than p65 at all of the concentrations tested. Theseresults demonstrated that AOP down-regulated MMP-2/-9 expres-sion through transcriptional down-regulations of NF-jB.

4. Discussion

Mollusks represent a valuable source of new compounds andare rich sources of structurally diverse bioactive nitrogenous com-ponents (Aneiros & Anoland, 2004; Pádraigín & Richard, 2012). Pre-vious studies have reported that bioactive peptides may be isolatedfrom mollusks, based on emerging evidence of potential healthbenefits, activities including ACE inhibitory, anticancer, antihyper-tensive and antioxidant effects, (Je, Park, Jung, Park, & Kim, 2005;Rajapakse, Mendis, Byun, & Kim, 2005; Tsai et al., 2008; Wesson& Hamann, 1996). The actual effect of these peptides is determinedby their amino acid composition and sequence levels. Interestingly,bioactive peptides from marine animals have opened a new per-spective for pharmaceutical developments and may fight againstagainst multiple tumour types. The discovery of novel naturalproducts to block cancer growth and migration are the key goalsof cancer researchers. Consequently, we consider the possibilitythat bioactive peptide isolated from abalone (H. discus hannai) haveanticancer effects by inhibition of MMP expression. Our resultsdemonstrate that AOP inhibits MMP-2/-9 enzyme activity inHT1080 cells.

MMPs are principal enzymes in extracellular matrix degrada-tion, which is essential in the invasive growth, metastasis and angi-ogenesis of cancer and various chronic inflammatory diseases (Hu,Steen, Sang, & Opdenakker, 2007; Lee, Hwang, Choi, & Jeong, 2008).MMP-2/-9 are known to be involved in processes such as tumourinvasion and metastasis. Development of MMP inhibitors is of cur-rent interest as a new class of therapeutic cancer targets. Most ofall, fibroblasts play a vital role in wound healing processes by pro-ducing MMP-2/-9, and induce the pathogenesis. In the presentstudy, we selected HT1080 cells because they produce bothMMP-2/-9 enzymes, and are commonly used as a model systemto study several MMP-activities and expressions (Brooks & Schum-acher, 2001, chap. 12). Therefore, we have purified the bioactivepeptides as a strong and selective inhibitor of MMP-2/-9 from gas-trointestinal digests of abalone intestine. Qian et al. (2012) re-searched the anti-inflammatory effects of abalone intestine on.We firstly prepared in vitro gastrointestinal digests of abaloneintestine, from which bioactive peptides could be released andmodified from the gastrointestinal digestion. The abalone intestinegastrointestinal digests were below 10 kDa molecular weight,which was fractionated using UF membrane bioreactor systems.Many researchers report that low molecular weight peptides aremore potent as bioactive peptides (Chen, Suetsuna, & Yamauchi,1995; Rajapakse et al., 2005). Gelatin zymographic analysis exhib-ited down-regulation of MMP-2/-9 in the presence of fractions. Inthis study, we demonstrated that the FrV of purification from aba-lone intestine gastrointestinal digests (below 10 kDa) significantlysuppressed MMP-2/-9 activities with PMA-stimulated (10 ng/ml),compared with the blank group (Fig. 1C). This active fraction wasfurther separated by RP-HPLC (Fig. 2A). Several studies have ob-served that peptides that are proline-rich and/or leucine-richmay possess ACE inhibitory (Byun & Kim, 2001), antimicrobial(Gueguen et al., 2009; Klara et al., 2008; Pavlina et al., 2011), anti-oxidant (Kim, Je, & Kim, 2007; Mendis, Rajapakse, & Kim, 2005),antitumor (Masayoshi, Yoshi, Mayumi, Andi, & Motomasa, 2012;Pettit et al., 1993), cytotoxic (Rana, Khalil, Valerie, & Hendrik,

Fig. 2. (A) The fraction V, exhibiting the highest inhibitory effect on gelatin activity, was applied onto a Shodex OHpack SB 803 HQ (8–300 mm) column with a buffercontaining 0.01% TFA at flow rate of 0.7 ml/min. The complete course was monitored using a UV detector at 215 nm. (B) Gelatin zymography for the determination of MMP-2/-9 activities in fractions of AIGID III treated HT1080 cell line. (C) Areas and relative intensities of gelatin-digested bands by MMP-2/-9. The results presented are themean ± S.D. of a triplicate experiment. P < 0.05; P < 0.01. (D) Identification of the molecular mass and amino acid sequence of peptide (fraction V-2) using ESI/MSspectroscopy. (E) Chemical structure of the AOP.

V.-T. Nguyen et al. / Food Chemistry 141 (2013) 503–509 507

Fig. 3. Effects of FrV-2 (the purified AOP) on the viability of HT1080 cells in thepresence and absence of FBS. Cells were treated with the purified AOP at 10, 50 and100 lM. The cell viability was determined by MTT assay after 36 h. Results ofindependent experiments were averaged and represented as percentage cellviability. The results presented are the mean ± S.D. of a triplicate experiment.

P < 0.05; P < 0.01.

Fig. 4. (A) Effect of the purified AOP on protein expressions of MMP-2/-9 in HT1080cells stimulated with PMA (10 ng/ml). Cells were pretreated with differentconcentrations of peptide for 1 h and PMA incubation was then continued for36 h. The protein expression levels were detected using Western blot analysis. b-Actin was used as an internal standard for normalisation of MMP-2/-9 proteins. (B)Areas and intensities of protein bands were determined by densitometry andexpressed as a percentage MMP expression compared to protein levels of PMA-alone treated cells. The results presented are the mean ± S.D. of a triplicateexperiment. P < 0.05; P < 0.01.

Fig. 5. (A) Effect of the purified AOP on protein expressions of p50 and p65 inHT1080 cells stimulated with PMA (10 ng/ml). Cells were pretreated with differentconcentrations of peptide for 1 h and PMA incubation was then continued for 36 h.Equal amounts of protein in the cell lysates were electrophoresed and p50 and p65protein levels in cytosol were determined using specific antibodies. b-Actin wasused as an internal standard for normalisation of p65 and p50 protein. (B) Areas andintensities of protein bands were determined by densitometry and expressed as apercentage NF-jB expression compared to protein levels of PMA-alone treated cells.The results presented are the mean ± S.D. of a triplicate experiment. P < 0.05;

P < 0.01.

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2011) and HIV-1 protease-inhibiting activity (Lee & Maruyama,1998). In addition, the MMP-2/-9 activities were reduced in thepresence of proline (Roomi, Ivanov, Niedzwiecki, & Rath, 2004).Hence, the proline-rich AOP, novel peptide from abalone (H. discushannai) inhibits MMP-2/-9 expression. Through ESI/MS spectros-

copy, the AOP was identified as Ala-Glu-Leu-Pro-Ser-Leu-Pro-Gly(MW = 782.4 Da) (Fig. 2D). The AOP did not exert any cytotoxic ef-fect on HT1080 cells (Fig. 3). In Western blot analysis, the expres-sion levels of MMP-2/-9 were dose-dependently inhibited bytreatment with the AOP without any cytotoxic influence (Fig. 4).Peptide suppressed the expression and secretion of MMP-2/-9 bydown regulation of MMP-2/-9 protein levels. However, the proteinexpression level of MMP-2 was higher than that of MMP-9 inHT1080 cells.

Furthermore, NF-jB activity suppresses the tumor growth andmetastasis of human cancer HT1080 cells due to the inhibition ofMMP-2/-9 (Mendis, Kim, Rajapakse, & Kim, 2009). Down-regula-tion of NF-jB resulted in production of low levels of both p50and p65 proteins and directly affected the activation process ofMMP-2/-9 expression (Rajapakse et al., 2007). The promoter regionof the MMP-2/-9 gene contains binding sites for NF-jB, we suggestthat the AOP was involved in suppression of NF-jB protein expres-sion and presumed inhibition of MMP-2/-9 with the PMA-stimu-lated expressions (Fig. 5).

5. Conclusion

The results obtained in the present study showed that fractionsof abalone intestine gastrointestinal digests, including FrI–FrVII,were purified from abalone (H. discus hannai). Among the purifiedfractions, FrV exhibited strong MMP-2/-9 inhibitory activity. Q-TOFmass spectrometer revealed the amino acid sequence of the novelpeptide (Ala-Glu-Leu-Pro-Ser-Leu-Pro-Gly) and molecular mass

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(MW = 782.4 Da). Furthermore, we demonstrated significant inhi-bition of MMP-2/-9 by the AOP in HT1080 cells. Additionally, theAOP may be anticancer active through down-regulations of tran-scription factors NF-jB. Thus, the AOP is a natural bioresourcesthat may possibly be used as a valuable chemopreventive agentor food supplement for reducing cancer risk.

Acknowledgements

This study was supported by Fishery Commercialization Tech-nology Development Program, Ministry for Food, Agriculture, For-estry and Fisheries, Republic of Korea, and a Grant from the It wasalso supported by Wando-County Program for R&D Services, Wan-do-gun, Jeonnam, Republic of Korea through the Program for theRegional Innovation System (RIS). It was also supported by Wan-do-County Program for R&D Services, Wando-gun, Jeonnam,Republic of Korea.

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