A novel peptide derived from Haliotis discus hannai inhibits the migration of MKN-28 gastric cancer cells through downregulation of [beta]-catenin signaling.
KEY WORDS: abolone, Haliotis discus hannai, [beta]-catenin signaling, Cdc42, gastric cancer, Racl
Recent studies have focused on bioactive compounds isolated from marine organisms to treat diseases such as leukemia, autoimmune diseases, and cancer (Hamed et al. 2015, Hansen & Andersen 2016). The abalone (Haliotis discus hannai Ino) is an edible shellfish, large marine gastropod molluscs from the Haliotidae family (Lee 2004). The meat of abalone is considered a delicacy in Asia, especially, in Korea, China, and Japan, which has become a commercially important food resource (Kresge et al. 2001, Lee 2004). Although previous studies have proved that abalone presents some bioactivities such as antioxidant, antistress, immunomodulating, and anticancer activities (Zhu et al. 2008, Sun et al. 2010, Da Yong et al. 2011), antimetastatic effects of abalone have not been investigated.
Gastric cancer is one of the most common diseases in the world, and the incidence of gastric cancer is the highest in men in northeast Asia (Korea, China, and Japan) (Hartgrink et al. 2009). Moreover, the prognosis for gastric cancer remains poor due to extensive metastatic characteristics (Oue et al. 2005). As metastasis (formation of the secondary tumor at a distant site from their primary site) involves complex and sequential molecular mechanisms such as migration, invasion, intravasation, extravasation, and colonization, it is considered a serious impediment to the treatment of gastric cancer (Gupta & Massague 2006, Steeg 2006, Klein 2008). [beta]-catenin is the principal component of adherens junctions at cell-cell contact and is anchored to the cytoskeleton to support cellular architecture and organization of the cytoskeleton. [beta]-catenin is also a principal component in the Wnt signaling pathway implicated in tumor promotion (Polakis 2000, Logan & Nusse 2004, Kam & Quaranta 2009). Cytosolic [beta]-catenin is phosphorylated by glycogen-synthase kinase 3[beta] (GSK-3[beta]) involved in the adenomatous polyposis coli complex and subsequently degraded through ubiquitin-proteasome pathway in the absence of Wnt (Polakis 2000, Tian et al. 2011). On the other hand, in the presence of Wnt, GSK-3[beta] activity is prevented by activated Wnt signaling. Thus, [beta]-catenin accumulates in the cytoplasm, translocates to the nucleus, and binds to transcription factors such as T-cell factor and lymphoid enhancer-binding factor (Polakis 2000, Logan & Nusse 2004). Consequently, [beta]-catenin target genes are expressed to induce cell proliferation and cell differentiation (Cavallaro & Christofori 2004).
Cancer cells must gain motility for the migration of cancer away from the primary cancer site. At this time, cells use two well-known cell membrane protrusions, lamellipodia and filopodia, to move into the surrounding extracellular matrix (Le Clainche & Carlier 2008, Micalizzi et al. 2010). A sheet-like protrusion lamellipodia and a rod-like protrusion filopodia are composed of actin cytoskeleton at the plasma membrane. Actin reorganization for formation of protrusions is controlled by many different signaling pathways through activities of Rashomologous GTPases (Rho GTPases), including Racl and Cdc42 (Nobes & Hall 1995, Yamaguchi & Condeelis 2007).
In this study, the effects of novel peptide derived from abalone (Haliotis discus hannai) on cancer cell migration were investigated using MKN-28 gastric cancer cells. Subsequently, it was examined whether the effects of this peptide on cell migration were connected to the downregulation of [beta]-catenin signaling and the disorganization of cellular protrusions.
MATERIALS AND METHODS
Design of Novel Peptide A2 and its Physical Properties
A novel peptide A2 was derived from a propeptide purified from the gill of the abalone, Haliotis discus hannai. The parent peptide that comprises 47 amino acids had an antimicrobial effect (unpublished data, Professor Park, Pukyong National University). To design A2, both a-helical structure and C-terminal amination were considered because of its optimal amphipathic characteristics that increase its net positive charge and stability. The secondary structure was predicted by the Gamier- Osguthorpe-Robson method for selection of the optimal a-helical region in the parent peptide. A Schiffer Edmundson plot was obtained to optimize amphipathic characteristics, and sequence length was modified using DNASIS v2.5 demo program (Hitachi Software Engineering Co. Ltd., Japan). Hydrophobicity and hydrophobic moment were also obtained using this program. Isoelectric point and molecular weight were measured by ExPASy (Hitachi Software Engineering Co. Ltd.). The physical properties of A2 are obtained from the various methods mentioned previously. The peptide A2 is an [alpha]-helical amphipathic structure composed of 14 amino acids, and has a positive net charge (+5), high hydrophobicity, and hydrophobic moment. The calculated molecular weight is 1,466.9 Da.
To synthesize A2, a solid-phase synthesis method was conducted by ASP48S PepSynthesizer (Peptron Inc., Daejeon-si, Korea) using 9-fluorenylmethoxycarbonyl (Fmoc) polypeptide active ester chemistry. Two forms of the peptide, one amidated (N[H.sub.2]) and the other with a free carboxy terminus (COOH), were synthesized with Fmoc-NH-SAL resin or Fmoc-resin. The synthetic peptide was purified using Vydac Everest C18 column (10 [micro]m, 300 [Angstrom], 22 x 250 mm) with a water-acetonitrile linear gradient of 3%-40% acetonitrile in 0.1% trifluoroacetic acid. Molecular weights and purities (>95%) of the synthetic peptides were examined using LC/ MS (Agilent HP1100 series; Agilent Technologies, Santa Clara, CA) and reversed-phase HPLC with CapCell-Pak C18 reversed-phase column (5 [micro]m, 300 [Angstrom], 4.6 x 250 mm; Shiseido, Tokyo, Japan) (molecular weights: 1,466.9 Da and formula: [C.sub.71][H.sub.123][N.sub.19][O.sub.14]).
Human gastric cancer cell line MKN-28 cells (Korean Cell Line Bank, Seoul-si, Korea) were cultured in RPMI 1640 medium (Corning, Manassas, VA) supplemented with 10% fetal bovine serum (FBS; Corning, Manassas, VA) and 1% antibiotic solution (100 U/ml penicillin and 100 mg/ml streptomycin; PAA Laboratories, GmbH, Austria). Human embryonic kidney (HEK-293) cells (American Type Culture Collection, Manassas, VA) were cultured in DMEM medium (Corning) supplemented with 10% FBS and 1% antibiotic solution. Human umbilical vein endothelial cells (HUVEC; American Type Culture Collection) were cultured in EGM-2 BulletKit (Lonza, Walkersville, MD) containing 10% FBS and 1% antibiotic solution. All cell cultures were maintained in a humidified incubator with 5% C[O.sub.2] at 37[degrees]C.
Cell Viability Assay
MKN-28, HUVEC, or HEK-293 (1.0 x [10.sup.4] cells in each well) were plated on 96-well cell culture plates and incubated for 24 h. After incubation, the cells were exposed to various concentrations of A2 (0, 5, 10, 15, 20, and 25 [micro]M) and incubated for 24 h. WST-1 solution (Daeil Laboratory service, Seoul-si, Korea) was added to the medium and kept for 4 h, and the absorbance was measured at 460 nm with a microplate reader (Molecular Devices, Sunnyvale, CA).
MKN-28 cells were seeded in an Ibidi [beta]-dish (Ibidi GmbH, Germany) and incubated for 24 h, and then inserts were carefully detached to make a wound. The monolayer was washed with 1x phosphate-buffered saline (PBS) to remove debris or the detached cells from the monolayer, and then A2 was added. The cells were incubated at 37[degrees]C and photographed immediately and monitored at specific time (12, 18, or 24 h) using an inverted microscope (Olympus CKX41; Olympus, Shinjuku, Tokyo, Japan).
Transwell Migration Assay
The chemotactic motility of MKN-28 cells was determined using a transwell with 6.5-mm-diameter polycarbonate filters (8 [micro]m pore size); Corning, Tewksbury, MA). The lower chambers were filled with 20% FBS. MKN-28 cells (5 x [10.sup.4] cells/well) suspended in 200 pi of media without FBS were seeded in each transwell. The cells were treated with various concentrations of peptide A2 (5, 10, and 15 [micro]M) and allowed to migrate for 24 h. Nonmigrated cells were removed using cotton swabs and migrated cells were fixed with 4% formaldehyde solution (Junsei, Tokyo, Japan) and stained with 1% crystal violet (Sigma, Louis, M A). Images were taken using an inverted microscope (x100 magnification). To quantify migrated cells, the cells were lysed with 2% sodium dodecyl sulfate for 1 h and measured with a microplate reader at 570 nm. Three independent experiments were conducted for the transwell migration assay.
MKN-28 cells were washed with 1x PBS and collected by centrifugation. For immunoblotting, whole-cell lysates were lysed in ice-cold lysis buffer (iNtRONBiotechnology, Seongnam-si, Korea). After incubation on ice for 30 min, the debris was removed by centrifugation at 14,000 rpm for 20 min at 4[degrees]C. For nuclear extracts, cells were lysed in NE-PER extraction reagent (Pierce, Rockford, 1L) according to the manufacturer's protocol. Protein samples were resolved in 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were then transferred to a nitrocellulose membrane and blocked with 5% skim milk in 1x PBST buffer (135 [micro]M NaCl, 2.7 mM KCl, 4.3 mM NaP[O.sub.4], 1.4 mM K[H.sub.2]P[O.sub.4], and 0.5% Tween-20) for 1 h at room temperature. The blots were probed with the primary antibodies overnight at 4[degrees]C, then washed three times with 1x PBST. The blots were incubated with horseradish peroxidase-coupled anti-rabbit immunoglobulin G (IgG) or anti-mouse IgG as the secondary antibodies for 1 h at room temperature. The blots were washed in 1x PBST, and proteins were detected by an enhanced chemiluminescent (ECL) detection solution (AbFrontier, Seoul-si, Korea). All rabbit monoclonal antibodies, anti-[beta]-catenin, anti-p-[beta]-catenin Ser33/Ser37/Thr41, anti-[beta]-GSK-3[beta] Ser9, anti-c-Myc, anti-Cdc42, anti-Racl, anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-[beta]-actin, mouse monoclonal antibody anti-GSK-3[beta], anti-rabbit IgG, and anti-mouse IgG as second antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA). Mouse monoclonal antibody anti-Cyclin D1 was purchased by Abeam (Cambridge, United Kingdom). Quantification of the relative intensity of the bands was analyzed by Image J (http://rsb.info. nih.gov/ij/index.html) (National Institutes of Health, Bethesda, MD). [beta]-actin was used as controls for whole-cell lysates while GAPDH and histone H3 were used as controls for cytoplasmic and nuclear fractions, respectively.
MKN-28 cells were allowed to grow on glass coverslips for 24 h. After incubation, the cells were treated with peptide A2 for 24 h, and then the cells were washed with 1x PBS, stained with 1 [micro]g/ml diamidino-2-phenylindole (DAPI) (Roche Diagnostics GmbH, Mannheim, Germany) for 20 min at 37[degrees]C, fixed with 4% formaldehyde for 15 min at room temperature, and blocked with 1% bovine serum albumin in PBS. Cells were then incubated with anti-rabbit [beta]-catenin or Racl antibody overnight at 4[degrees]C and incubated with Alexa488-conjugated goat anti-rabbit IgG (Cell Signaling Technology Inc., Danvers, MA) for 1 h at room temperature. Coverslips were mounted onto microscope slides using ProLong Gold Antifade mounting medium (Life Technologies, Carlsbad, CA). Immunofluorescence was observed and photographed using a laser scanning confocal microscope (Carl Zeiss LSM 700; Carl Zeiss, Jena, Germany).
MKN-28 cells were washed twice with PBS and fixed in 4% formaldehyde for 10 min at room temperature. After additional washes with PBS, the cells were permeabilized with 0.1% Triton X-100 in 1x PBS for 5 min and washed again with PBS three times. To stain filamentous actin (F-actin), 0.2 [micro]g/ml phalloidin-tetramethylrhodamine isothiocyanate (Life Technologies) was used. Fluorescent dyes were diluted in blocking solution (1% bovine serum albumin in PBS) and added to the cells and kept for 40 min at 37[degrees]C, and then washed with 1x PBS. DAPI solution without methanol was added to stain nucleus for 20 min at 37[degrees]C. After washing three times with 1x PBS, coverslips were mounted on a microscopy slide with ProLong Gold Antifade mounting medium. F-actin cytoskeleton imaging was performed with laser scanning confocal microscope.
All data presented in this study were performed at least three times, independently in triplicate. The prism 6.0 software (GraphPad, La Jolla, CA) for Windows was applied to identify the statistical significant differences between experimental and control values. Data are presented as mean [+ or -] SD, and Dunnett's multiple comparisons test was performed; P < 0.05 was deemed statistically significant.
A2 Inhibits MKN-28 Gastric Cancer Cell Proliferation
To investigate effects of A2 on MKN-28 cell migration, first, an antiproliferative effect was assessed using MKN-28 cells, HEK-293 cells, and HUVEC at various concentrations of A2, where each cell line was treated with A2 for 24 h. Cell proliferation was significantly suppressed in MKN-28 cells at concentrations of 20 or 25 [micro]M, where P = 0.0004 and P = 0.0003, respectively. A2 did not exhibit a statistically significant decrease at concentrations of 5, 10, or 15 [micro]M, where P = 0.6633, P = 0.1761, and P = 0.0761, respectively (Fig. 1). When HUVEC (blood vessel endothelial cells) and HEK293 (kidney epithelial cells) were treated with A2, the cell proliferation of both cell lines was not decreased at the same concentrations of A2. Hence, to prevent the possibility of A2-induced cell death in both normal and cancer cells, a concentration range from 5 to 15 [micro]M A2 was chosen for further experiments.
A2 Inhibits the Cell Migration of MKN-28 Cells
The effects of A2 on cell migratory ability were measured when MKN-28 cells were treated with A2 as cancer cell migration is a key step during metastasis. As A2 did not show antiproliferative effect on MKN-28 cells at concentrations less than 15 [micro]M, A2 was added at final concentrations of 5, 10, and 15 [micro]M to conduct wound healing and transwell migration assays. In the wound healing assay, the wounded area was completely recovered after 24 h in control, whereas it was rarely recovered with the addition of 15 [micro]M A2. The wounded area was maintained at over 80% at 12 and 24 h (Fig. 2A) after treatment with A2. Equally, A2 impeded cancer cell migratory ability on transwells in a dose-dependent manner, where it significantly (P = 0.0003) decreased cell migration by approximately half at 15 [micro]M A2 (Fig. 2B). Taken together, these results indicate that A2 has the potential to regulate metastasis by inhibiting the migratory ability of gastric cancer cells.
A2 Controls the States of fi-Catenin and GSK-3$ in MKN-28 Cells
The loss of E-cadherin leads to the release of [beta]-catenin from adherens junctions to the cytoplasm and inactivates GSK-3[beta]. As shown in Figure 3A, B, the expression and phosphorylation levels of [beta]-catenin and GSK-3P were changed, but the expression level of GSK-3P did not change with A2 in a time- or dose-dependent manner. A low-level phosphorylation of [beta]-catenin was examined at time zero or in the control, which indicates that [beta]-catenin is present in an active state at all times. The phosphorylated [beta]-catenin, on the other hand, increased gradually in A2-treated cells (Fig. 3A, B), signifying that [beta]-catenin is targeted for ubiquitin-mediated protein degradation. Glycogen-synthase kinase 3[beta] is responsible for [beta]-catenin phosphorylation, which is subsequently targeted for proteasomal degradation (Polakis 2000). The phosphorylation state of GSK-3[beta] decreased to zero with increased time and dose (Fig. 3A, B), which indicates that A2 assists in the recovery of GSK-3[beta] activity as GSK-3P is inactivated by phosphorylation. These results indicate that A2 can control [beta]-catenin signaling through regulation of the phosphorylation status of [beta]-catenin and GSK-3[beta] in MKN-28 cells.
A2 Affects the Translocation of [beta]-Catenin in MKN-28 Cells
Nuclear [beta]-catenin functions as a transcriptional factor (Ng et al. 2005); therefore, it was examined whether A2 affects the distribution of [beta]-catenin in MKN-28 cells. Control cells exhibited the localization of [beta]-catenin in cytoplasm and nucleus (arrow), whereas in A2-treated cells, [beta]-catenin localization was predominantly localized at the cell-cell border (Fig. 4A, dashed arrow). The fact that the nuclear fraction of [beta]-catenin was decreased in response to A2 treatment in MKN-28 cells (Fig. 4B) supports that A2 inhibits the translocation of [beta]-catenin to the nucleus. To identify whether A2 can affect target genes of [beta]-catenin such as Cyclin D1 and c-Myc, the expression level of Cyclin D1 and c-Myc was examined. As shown in Figure 4C, the expression level of both proteins was reduced by half in MKN-28 cells over 24 h when treated with 15 [micro]M A2. Taken together, these results demonstrate that A2 can repress the state of [beta]-catenin, leading to the downregulation of Cyclin D1 and c-Myc in MKN-28 cells.
A2 Downregulates Rad and Cdc42 in MKN-28 Cells
To explore molecular mechanisms of A2, changes in expression level of Rho GTPases in the A2-treated cells were examined, as Rho GTPases, including Racl and Cdc42, induce cell migration in response to various stimuli (Nobes & Hall 1995). As shown in Figure 5A, B, the expression level of Racl and Cdc42 was decreased when MKN-28 cells were treated with A2 in a time- and dose-dependent manner. In addition, it was confirmed that A2 was able to alter the localization of Racl, because Racl was located at the edge of cells in the absence of A2 (white arrow). In contrast, in A2-treated MKN-28, Racl was generally diminished throughout cells and located away from the edge of cells (Fig. 5C). As Racl and Cdc42 are involved in the formation of lamellipodia and filopodia in moving cells (Wheeler & Ridley 2004), effects of A2 on the formation of lamellipodia and filopodia were investigated. The control showed actin-rich lamellipodia and filopodia formed normally at the leading edge of cells, whereas A2-treated cells exhibited reduced thickness of lamellipodia and defective construction of filopodia (Fig. 5D). Taken together, these results indicate that A2 disorganized lamellipodia and filopodia through suppression of the expression of Racl and Cdc42 in MKN-28 cells.
Abalone, including Haliotis discus, are largely cultivated and used as valuable food source in east Asian countries from ancient times. Recent studies have shown that substances isolated from abalone, such as glycoproteins, lipids, and peptides, have immune-stimulant, antiviral, and antioxidant activities (Lee 2004, Zhu et al. 2008, Sun et al. 2010, Da Yong et al. 2011). It has not been fully elucidated that peptides isolated from abalone have antitumor activity. To investigate anticancer effects of peptides, novel peptide A2 from abalones was synthesized; however, detailed physical characteristics of A2 were not fully identified.
Although a statistically significant inhibition in cell proliferation did not result for all three cell lines (HUVEC, HEK-293, and MKN-28 cells) at concentrations less than 15 [micro]M A2, a statistically significant inhibition of cell proliferation was only present in MKN-28 cells at concentrations greater than 20 [micro]M. These results suggest that A2 can decrease gastric cancer cell proliferation without cytotoxic effects on both noncancerous endothelial cell HUVEC and epithelial cell HEK-293 cells. In addition to its antiproliferative effects, A2 may impede cancer cell metastasis as A2 significantly inhibited cancer cell migration. Therefore, the molecular mechanisms of A2 inhibiting migration in MKN-28 cells were investigated.
Activation of Wnt/[beta]-catenin signaling is found in gastric cancer tissues and in different types of gastric cancer cell lines (Yokozaki 2000, Clements et al. 2002, Ooi et al. 2009), components of which are usually deregulated in gastric cancer in a variety of ways, including mutations and gain of function in positive and negative regulators (Chiurillo 2015). Hence, this study focused on the regulatory effects of A2 on [beta]-catenin signaling in MKN-28 cells. Although normally GSK-3P phosphorylates [beta]-catenin at Ser33/Ser37/Thr41, which is then targeted for ubiquitin-mediated protein degradation (Liu et al. 2002, Valenta et al. 2012), in the presence of Wnt, the activity of GSK-3[beta] is inhibited through phosphorylation at Ser9 by protein kinases, such as p70S6 kinase and Akt, which attenuates GSK-3[beta]-mediated phosphorylation of [beta]-catenin (Ali et al. 2001, Fang et al. 2007, Nakayama et al. 2009). Consequentially, nonphosphorylated [beta]-catenin is accumulated in the cytoplasm and translocated into the nucleus (Harris & Peifer 2005, Fang et al. 2007, Chiurillo 2015). In A2-treated MKN-28 cells, phosphorylation of [beta]-catenin at Ser33/Ser37/Thr41 was enhanced, whereas phosphorylation of GSK-3[beta] at Ser9 was reduced; hence, it was suggested that A2 can promote [beta]-catenin degradation via an increase in its phosphorylation after the recovery of GSK-3[beta] activity. Therefore, reduced expression level of [beta]-catenin in response to A2 treatment might be caused by downregulation of phosphorylated GSK-3[beta] at Ser9. Once the translocation of [beta]-catenin occurs, it forms a complex with transcriptional factors, T-cell factor and lymphoid enhancer-binding factor, which triggers the transcription of target genes related to gastric carcinogenesis including Cyclin D1 and c-Myc (He et al. 1998, Shtutman et al. 1999, Lin et al. 2000, Shan et al. 2009). Hence, it was investigated whether A2 can inhibit the localization of [beta]-catenin in MKN-28 cells. As expected, nuclear [beta]-catenin levels were reduced, and some [beta]-catenin were transferred at the cell-cell border in A2-treated cells. Moreover, the fact that [beta]-catenin target genes, Cyclin D1 and c-Myc, were downregulated by A2 treatment confirmed the hypothesis that A2 can regulate [beta]-catenin signaling. These results suggest that A2 can control [beta]-catenin signaling by extricating GSK-3[beta] from phosphorylation and the translocation of [beta]-catenin into nucleus, resulting in decreased expression levels of Cyclin D1 and c-Myc.
To migrate, cells use actin-rich membrane protrusions at the leading edge of the cell, such as lamellipodia and filopodia. Actin polymerization drives forward cell membrane protrusions, forming flat and sheet-like regions at the leading edge called lamellipodia, which are the central structures for cell locomotion. Finger-like extension filopodia emerge from the lamellipodia, which contain bundles of actin filaments, and penetrate into the surrounding extracellular matrix (Yamaguchi & Condeelis 2007, Le Clainche & Carlier 2008, Ridley 2011). According to a recent study, the destruction of cell membrane protrusion decreases cell motility leading to the decrease of cell migration (Cheng et al. 2013); therefore, it is suggested that A2 inhibits cancer cell migration by demolishing an extended actin-rich membrane protrusion, lamellipodia and filopodia, at the leading edge of MKN-28 cells. Both Racl and Cdc42 belonging to Rho GTPases are involved in the formation of cell membrane protrusions by transmitting signaling pathways, which coordinate with cell migration (Ridley 2011). These results suggest that the expression of Rac1 and Cdc42 was decreased in A2-treated MKN-28 cells and confirm that A2 can inhibit cell migration by disorganizing lamellipodia and filopodia at the leading edge of cells via suppression of expression of Rac1 and Cdc42. Figure 6 shows a proposed signaling pathways for A2 in which A2 inhibits [beta]-catenin signaling through enhancing GSK-3[beta] activity and disorganizes cellular protrusions through downregulation of Racl and Cdc42, leading to the inhibition of cancer cell migration.
In conclusion, with regard to the anticancer effects of the novel peptide A2 derived from abalone Haliotis discus hannai, to our knowledge it was the first report on the capabilities of A2 to control metastasis through its inhibitory effects on gastric cancer cell migration by disrupting [beta]-catenin signaling and disorganizing cellular protrusions at the leading edge of cells. These results also propose two different signaling pathways regulated by A2 treatment. One is the inhibition of cancer cell migration by inhibiting [beta]-catenin signaling through enhancing GSK-3[beta] activity. The other is the disorganization of lamellipodia and filopodia, thereby downregulation of Racl and Cdc42. Therefore, A2 may be developed as a metastasis suppressor for treating gastric cancer in the future.
This study was supported by 213004-04-4-SB820, Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA), and Korea Forest Service (KFS).
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NAN-HEE KIM, (1) CHANG-WON KANG, (1) MIN-SEOK PARK, (1) CHUL-WOONG OH, (2) YONG BAE SEO, (3) JONG KYU LEE, (1) JONG-MYOUNG KIM, (4) HAN KYU LIM (5) * AND GUN-DO KIM (1) *
(1) Department of Microbiology, College of Natural Sciences, Pukyong National University, Busan 48513, Republic of Korea; (2) Department of Marine Biology, College of Fisheries Sciences, Pukyong National University, Busan 48513, Republic of Korea; (3) Institute of Fisheries Science, College of Fisheries Sciences, Pukyong National University, Busan 48513, Republic of Korea; (4) Department of Fishery Biology, College of Fisheries Sciences, Pukyong National University, Busan 48513, Republic of Korea; (5) Department of Marine and Fisheries Resources, Mokpo National University, Muan 58554, Jeonnam, Republic of Korea
* Corresponding authors. E-mails: email@example.com and gundokim@ pknu.ac.kr
Please note: Some tables or figures were omitted from this article.
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|Author:||Kim, Nan-Hee; Kang, Chang-Won; Park, Min-Seok; Oh, Chul-Woong; Seo, Yong Bae; Lee, Jong Kyu; Kim, Jo|
|Publication:||Journal of Shellfish Research|
|Date:||Oct 1, 2016|
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