Title: Protein hydrolysates from tuna cooking juice inhibit cell growth

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Title:
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Protein hydrolysates from tuna cooking juice inhibit cell growth and induce
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apoptosis of human breast cancer cell line MCF-7
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Names of authors:
Chuan-Chuan Hunga,b; Yu-Hsuan Yanga; Pei-Feng Kuoa; and Kuo-Chiang Hsua,b*
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Affiliation and address of authors:
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a
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Department of Nutrition, China Medical University, No. 91, Hsueh-Shih Road,
Taichung 40402, Taiwan.
b
Department of Health and Nutrition Biotechnology, Asia University, No. 500,
Lioufeng Road., Taichung 41354, Taiwan.
Short title:
Peptides induce apoptosis of MCF-7
*Corresponding author
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Tel.: +886 4 22053366 ext 7522; fax: +886 4 22062891
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E-mail address:[email protected] (K. C. Hsu)
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Abstract
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The effects of peptides from tuna cooking juice hydrolysates by Protease XXIII
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(PA) on cell growth and induction of apoptosis of human breast cancer cell line
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MCF-7 were determined. The PA hydrolysates showed antiproliferative activities up
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to 25% against MCF-7 cells, and the >2.5 kDa ultrafiltration fraction (PAH2.5)
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possessed the highest antiproliferative activity with an IC50 value of 1.39 mg/mL.
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PAH2.5 induced cell cycle arrest in S phase through the increases of p21 and p27, and
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decrease of cyclin A expression. Further, PAH2.5 also induced apoptosis of MCF-7
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cells by downregulation of the expression of Bcl-2, PARP and caspase 9, and
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upregulation of the expression of p53, Bax and cleaved capase 3. Two peptides were
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identified in PAH2.5 as KPEGMDPPLSEPEDRRDGAAGPK (2449.292 Da) and
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KLPPLLLAKLLMSGKLLAEPCTGR (2562.405 Da). Thus, tuna cooking juice may
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be a good protein source of antiproliferative peptides against MCF-7.
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Keywords: tuna cooking juice; peptides; breast cancer; antiproliferation; cell cycle;
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apoptosis
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1. Introduction
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Breast cancer is now the most common cause of female cancer and leading cause
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of cancer deaths among women in the United States and many other parts of the world
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(Ferlay, Shin, Bray, Forman, Mathers, & Parkin, 2010; Jemal, Siegel, Xu, & Ward,
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2010). In Taiwan, breast cancer is the most leading incidence and the 4th cause of
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death from female cancer, and, in recent years, its mortality rate has increased and
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average age of death decreased.
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Tuna is one of the most important fisheries in Taiwan, and its production and
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output value per year are over 300,000 tonnes and 31 billion NT dollars
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(approximately US$ 1 billion), respectively. Tuna cooking juice, a byproduct during
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the processing of canned tuna, contains approximately 5% proteins containing about
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30% hydrophobic amino acids (Jao, & Ko, 2002; Huang, Jao, Ho, & Hsu, 2012) and
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is always discarded. Our research group has determined that tuna cooking juice
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possessed some physiological functions, such as antioxidative (Hsu, Lu, & Jao, 2009),
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antihypertensive (Hsu, Cheng, & Hwang, 2007) and dipeptidyl peptidase IV (DDP-IV)
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inhibitory (Huang et al., 2012) activities.
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Peptides derived from various protein sources were also investigated to show
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antitumour or antiproliferative activities against cancer cells. Two peptides
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(dermaseptins B2 and B3) in the skin secretions of the South American tree frog
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inhibited the proliferation of the human prostatic adenocarcinoma PC-3 cell line with
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an EC50 of 2-3 μM (van Zoggel et al., 2012). FF/CAP18, an analogue peptide derived
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from an endogenous cathelicidin family member, showed antiproliferative activity
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against colon cancer cell line HCT116 with the loss of mitochondrial membrane
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potential, and resulted in the early stage of apoptosis (Kuroda et al., 2012). Lunasin
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from
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oncogene-induced transformation of mammalian cells and inhibit skin carcinogens in
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mice (Galvez, Chen, Macasieb, & de Lumen, 2001; Jeong, Jeong, Kim, & de Lumen,
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2007). A hydrophobic peptide, X-MLPSYSPY (1,157 Da) from defatted soy protein
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hydrolyzed with thermoase showed in vitro cytotoxicity on mouse monocyte
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macrophage cell line (Kim, Kim, Kim, Kang, Woo, & Lee, 2000).
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previous study, two hydrophobic peptides, LPHVLTPEAGAT (1,206 Da) and
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PTAEGGVYMVT (1,124 Da) isolated from tuna dark muscle byproduct had a
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dose-dependent inhibition effect on human breast cancer cell line MCF-7 (Hsu,
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Li-Chan, & Jao, 2011). A study has revealed that modulation of hydrophobicity of
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peptides plays a crucial role against cancer cells (Huang, Wang, Wang, Liu, & Chen,
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2011). Therefore, protein source with high contents of hydrophobic amino acids may
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have the potential to possess anticancer and antiproliferative activities against cancer
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cells. On the other hand, peptides were reported to be able to induce apoptosis in
soybeans,
was
found
to
suppress
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chemical
carcinogen
and
viral
In our
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tumour cells and as prime candidates for the development of anticancer therapeutics
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(Bhutia & Maiti, 2008).
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It is surprising that there were only few related research reports on
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antiproliferative and apoptosis of cancer cell lines induced by peptides obtained from
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food proteins, especially fish proteins. In this study, we tried to use commercial
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proteases, Protease XXIII (PA) to hydrolyze tuna cooking juice and then identify the
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antiproliferative activity on the human breast cancer cell line MCF-7. In addition, we
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investigated the effects of the hydrolysates on cell cycle and apoptosis of MCF-7, and
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the amino acid sequences of the peptides in the hydrolysates were also identified.
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2. Materials and methods
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2.1. Sample preparation
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A canned tuna processor in Chiayi County (Taiwan) supplied the tuna cooking
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juice in which the protein content was 4.71% (data not shown). The whole tuna fish
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(Thunnus tonggol) was cooked by steam (100-105℃) for 3-4 h, after which, the hot
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collected cooking juice was sealed in 400 mL polyethylene bags and then transferred
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to our laboratory immediately, and stored at 4℃ overnight. The cooking juice was
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filtrated through two layers of gauze to remove floating fats and solids, and the filtrate
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was collected and stored at -20℃ until used within 3 months.
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2.2. Chemicals and reagents
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Protease XXIII (PA) (specific activity of 3.8 units/mg solid), an endopeptidase
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prepared from Aspergillus melleus, was obtained in dry powder form from
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Sigma-Aldrich, Inc. (St. Louis, MO, USA). 3-(4,5-Dimethylthiazol-2-yl)-diphenyl
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tetrazolium
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2,4,6-trinitrobenzenesulphonic acid (TNBS) were purchased from Sigma-Aldrich, Inc.
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Other chemicals and reagents used were analytical grade and commercially available.
bromide
(MTT),
L-glutamine,
L-leucine
and
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2.3. Enzymatic hydrolysis
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Twenty-five millilitres of tuna cooking juice were adjusted to pH 7.5 by using 2
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M NaOH and then incubated at 37℃ in a water bath for 20 min prior to enzymatic
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hydrolysis. The hydrolysis reaction was started by the addition of enzyme solutions at
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the enzyme/substrate (E/S) ratio of 2.1% (25 mg enzyme powder dissolved in 1 mL
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ddH2O). After hydrolysis up to 6 h, the hydrolysate solutions were heated in a boiling
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water for 15 min to inactivate enzymes and then cooled in cold water at room
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temperature for 20 min. Hydrolysates were adjusted to pH 7.0 with 2 M NaOH and
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centrifuged (Centrifuge 05P-21, Hitachi Ltd., Katsuda, Japan) at 10,000g and 4℃ for
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10 min. The supernatant was lyophilised and stored at -20℃.
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2.4. Degree of hydrolysis
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The degree of hydrolysis (DH) of the hydrolysate was determined as the ratio of
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the amount of α-amino acid released during hydrolysis to the maximum amount of
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α-amino acid in tuna cooking juice (Benjakul & Morrissey, 1997). Properly diluted
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samples (125 μL) were mixed with 2 mL of 0.2125 M sodium phosphate buffer (pH
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8.2), followed by the addition of 1 mL of 0.01% TNBS. The mixtures were then
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incubated in a water bath at 50℃ for 30 min in the dark. The reaction was terminated
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by the addition of 2 mL of 0.1 M sodium sulphite. The mixtures were cooled down at
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ambient temperature for 20 min. The maximum amount of α-amino acid in tuna
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cooking juice was obtained by acid hydrolysis with 6 M HCl at 105℃ for 24 h. The
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acid-hydrolysed sample was then filtered through Whatman filter paper No. 1 to
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remove the unhydrolysed debris. The supernatant was neutralised with 6 M NaOH
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before α-amino acid determination. The absorbance was measured at 420 nm and
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α-amino acid was expressed in terms of L-leucine. The DH was calculated as follows:
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DH (%) = [(Lt-Lo)/(Lmax-Lo)] x 100, where Lt is the amount of α-amino acid released at
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time t; Lo is the amount of α-amino acid in original tuna cooking juice; Lmax is the
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maximum amount of α-amino acid in tuna cooking juice (Beak & Cadwallader,
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1995).
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2.5. Cell culture
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Human breast cancer MCF-7 cells and MCF-10A mammary epithelial cells
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purchased from Bioresource Collection and Research Center (BCRC) (Hsinchu,
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Taiwan) were cultured in a 37℃ humidified atmosphere with 5% CO2 in DMEM,
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supplemented with 10% FBS, 1% PSN and 1.5 g/L sodium bicarbonate (pH 7.1-7.2).
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2.6. MTT assay
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To avoid pH variation of the cell culture medium during sample solubilisation,
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fish hydrolysate stock solution was prepared in 0.1 M PBS (pH 7.4). The cells were
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seeded in a 96-well microtiter plate (1 x 104 cells/well) overnight, and then treated
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with various concentrations of hydrolysates and their ultrafiltration fractions. After
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incubating for 72 h, the effect of hydrolysates on cell growth was examined by the
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MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide) assay. About 20
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μL of MTT solution (5 mg/mL, Sigma Chemical Co.) were added to each well and
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incubated at 37℃ for 4 h. The supernatant was aspirated and the MTT-formazan
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crystals formed by metabolically viable cells were dissolved in 200 μL of isopropanol.
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Finally, the absorbance was read at 570 nm with a microplate reader. The hydrolysate
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concentration which gives 50 % growth inhibition is referred to as the IC50.
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2.7. Ultrafiltration (UF)
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The peptides of the hydrolysates were fractionated by ultrafiltration (model
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ABL085, Lian Sheng Tech. Co., Taichung, Taiwan) with spiral wound membranes
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having molecular mass cutoffs of 2.5 and 1 kDa. The fractions were collected as
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follows: >2.5 kDa, peptides retained without passing through 2.5 kDa membrane;
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1-2.5 kDa, peptides permeating through the 2.5 kDa membrane but not the 1 kDa
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membrane; <1 kDa, peptides permeating through the 1 kDa membrane. All fractions
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collected were lyophilized and stored in a desiccator until use.
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2.8. Cell Cycle
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For cell cycle analysis, cells were seeded at a density of 1×105 cells/well in
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6-well plates, cultured overnight, and then treated with various concentrations of PA
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hydrolysates. To analyze the cell cycle, after 72 h of treatment the cells were
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harvested by trypsinisation, washed in PBS, and fixed in 70% ice cold ethanol. The
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cell pellets were resuspended in 500 μL of a solution containing 50 μg/mL propidium
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iodide, 0.4 mg/mL RNase A, 0.1% Triton-X-100 in PBS buffer, and then incubated at
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37℃ for 30 min. The stained cells were subjected to DNA content/cell cycle analysis
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using an LSR flow cytometer.
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2.9. Apoptosis
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For apoptosis, the FITC Annexin V Apoptosis Detection Kit (BD Pharmingen TM,
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San Jose, CA, USA) was used to assess annexin V-positive cells. Wash cells twice
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with cold PBS and then resuspend cells in 1X annexin binding buffer. One hundred
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microliters of the solution was transferred to a 5 mL culture tube and add 5 μL of
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FITC Annexin V and 5 μL PI. Gently vortex the cells and incubate for 15 min at room
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temperature in the dark. After incubation, 400 μL of 1X annexin binding buffer were
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added to each tube and the cells were analyzed by flow cytometry using an LSR flow
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cytometer (BD Biosciences Inc., Franklin Lakes, NJ, USA).
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2.10 Identification of amino acid sequence by MALDI-TOF/TOF MS/MS
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The purified peptides were analyzed by matrix-assisted laser desorption
180
ionization time-of-flight mass spectrometry (MALDI-TOF MS), using a delayed
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extraction source and a 335 nm pulsed nitrogen laser. This analysis was carried out
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using a MALDI-TOF/TOF (UltraFlexIII, Bruker Daltonics Inc., Billerica, MA, USA).
183
Peptides
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α-cyano-4-hydroxycinnamic acid, and a droplet of the resulting solution was placed
185
on the sample target mass spectrometer. The droplet was dried by evaporation at room
solution
(0.6
μL)
was
mixed
10
with
0.6
μL
of
saturated
186
temperature and then loaded into the mass spectrometer for analysis. The instrument
187
was operated in positive ion reflection mode with the source voltage set at 20 kV. All
188
spectra were the results of signal averaging of 200 shots. Measurements were
189
determined in the mass range m/z 200-4000 Da, while the peptide sequencing was
190
determined by MS/MS spectra processing, using BioTools (Version 3.2; Bruker
191
Daltonics Inc., Billerica, MA, USA).
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2.11 Statistical analysis
Each data point represents the mean of three samples was subjected to analysis
195
of variance (ANOVA) followed by Duncan’s multiple range test, and the significance
196
level of P<0.05 was employed.
197
198
3. Results and discussion
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3.1. Degree of hydrolysis
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The DH of tuna cooking juice hydrolyzed with PA increased dramatically during
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the initial 1 h and 2 h, and increased gradually thereafter (Fig. 1A). The highest DH
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(%) of PA hydrolysates was 14.0% after the 6-h hydrolysis. The trend and curve
203
shape of the hydrolysis are similar to those reported in our previous studies (Hsu et al.,
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2009, 2011; Huang et al., 2012) and also to other studies on enzymatic hydrolysis of
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various protein sources (Bougatef et al., 2010; Dong, Zeng, Wang, Liu, Zhao, & Yang,
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2008; Klompong, Benjakul, Kantachote, & Shahidi, 2007).
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3.2. Antiproliferative activities of hydrolyates
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In our preliminary test, PA hydrolysates showed greater antiproliferative activity
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than the other two hydrolysates obtained from the hydrolysis by orientase 90N
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(Hankyu Bioindustry Co., Osaka, Japan) and alcalase (Novozymes North America
212
Inc., Salem, NC, USA). In the present study, therefore, only PA hydrolysates were
213
used for the further determinations.
214
The antiproliferative effect of the hydrolysates (concentration of 1 mg/mL)
215
derived from tuna cooking juice on breast cancer cell, MCF-7, after incubation for 72
216
h was investigated. As depicted in Fig. 1B, all the PA hydrolysates during 1-6 h
217
hydrolysis possessed significant antiproliferative activity as compared to the control
218
(p<0.05). Stronger antiproliferative activities (22-25%) were observed in PA
219
hydrolysates for 1, 2 and 4 h, but there were no significant differences between those
220
of the 3 hydrolysates (p>0.05). No correlation exhibited between degree of hydrolysis
221
and antiproliferative activity in this study, and this might imply the antiproliferative
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peptides were independent of molecular weight (Picot et al., 2006). For further
223
purification and investigation, PA hydrolysate for 1-h hydrolysis (PAH) was chosen
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224
based on time saving principle.
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226
3.3. Antiproliferative activity of UF fractions of hydrolysates
227
The antiproliferative activities of the UF fractions (>2.5 kDa, 1-2.5 kDa, and <1
228
kDa) of PAH are shown in Fig. 2. The peptides within the >2.5 kDa UF fraction
229
(PAH2.5) had the greatest antiproliferative activity of 37.8% (P<0.05), whereas those
230
within the <1 kDa and 1-2.5 kDa UF fractions (PAH1 and PAH1-2.5) displayed the
231
inhibition rates of only 20.0 and 22.8%, respectively (Fig. 2A). To the best of our
232
knowledge, the antiproliferative peptides derived from fish protein sources were
233
reported to have molecular weight (MW) of 440.9 Da from anchovy sauce (Lee, Kim,
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Lee, Kim, & Lee, 2003; Lee, Lee, Kim, Kim, & Lee, 2004) and those of 1,206 and
235
1,124 Da from tuna dark muscle (Hsu et al., 2011). The MWs of the antiproliferative
236
peptides obtained from soy protein and algae protein wastes were 1,157 and 1,309 Da,
237
respectively (Kim et al., 2000; Sheih, Fang, Wu, & Lin, 2010). However, some
238
studies reported the MWs of the antiproliferative peptides derived from various
239
sources to be greater than 1,400 Da. An antifungal peptide with MW of approximately
240
3.9 kDa, isolated from buckwheat seeds, possessed antiproliferative activities against
241
leukaemia (L1210), breast (MCF-7), liver embryonic (WRL68) and liver (HepG2)
242
cancer cells (Leung & Ng, 2007); and lunasin, a cancer-preventive peptide with MW
13
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of about 5.45 kDa, isolated from soy and barley, has been demonstrated to be
244
effective against chemical carcinogens and oncogenes in mammalian cells and in a
245
skin cancer mouse model (de Lumen, 2005). These findings demonstrate that there is
246
no correlation between antiproliferative activity and MW of peptides.
247
Fig. 2B shows the antiproliferative activity of PAH2.5 at various concentrations
248
(0.5-5 mg/mL). The inhibition rates ranged from 30.9 to 81.4% in a dose-dependent
249
manner; and the IC50 value against MCF-7 of PAH2.5 was 1.39 mg/mL. The peptides
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within the PAH2.5 at the concentrations between 1 and 10 mg/mL did not show any
251
cytotoxic effect on the cell viability of MCF-10A mammary epithelial cells (Fig. S1).
252
This result is similar to the study reported that the bitter melon extract inhibited breast
253
cancer cell line MCF-7 and did not show any cytotoxic effect on human primary
254
mammary epithelial cells, HMEC (Ray, Raychoudhuri, Steele, & Nerurkar, 2010).
255
The IC50 value of PAH2.5 in this study was lower than Huaier aqueous extract (IC50 =
256
4 mg/mL) (Zhang, Kong, Yan, Yuan, & Yang, 2010), therefore, the results indicated
257
that the PA hydrolysates would be a good source for the preparation antiproliferative
258
peptides.
259
260
261
3.4. Cell cycle
As the regulation of cell cycle is critical for the growth and development of
14
262
cancer, we determined the effect of PAH2.5 on cell cycle progression. The result
263
shown in Fig. 3A indicates that the treatment of PAH2.5 (concentrations of 0, 0.5, 1
264
and 1.5 mg/mL) with MCF-7 for a total of 72 h caused a concentration-dependent
265
accumulation of cells in the S phase, and a corresponding decrease in G0/G1 and
266
G2/M-phase fractions. As summarized in Fig. 3B, the cells in the S phase increased
267
by 21.79, 37.20, 41.38 and 47.96 % at concentrations of 0, 0.5, 1 and 1.5 mg/mL of
268
PAH2.5, respectively. Moreover, this correlated with the decreased population in
269
G0/G1, 71.87, 59.43, 54.75 and 50.22 %, and in G2/M, 6.33, 4.41, 3.27 and 5.44 %,
270
respectively. These results revealed that the PAH2.5 induced cell cycle of MCF-7
271
arrest in S phase.
272
To evaluate the role of cell cycle-regulating proteins in the MCF-7 treated with
273
PAH2.5, proteins were extracted from the PAH2.5-treated cells at 72 h for western blot
274
analysis. When compared with the control group, the expression of cyclin A was
275
significantly decreased in a concentration-dependent manner for the PAH2.5 treatment;
276
whereas the expressions of p21 and p27 were increased at 0.5 and 1 mg/mL, but
277
decreased at 1.5 mg/mL (Fig. 4). These results suggest that PAH2.5 induces cell cycle
278
arrest in S phase through increases of p21 and p27 proteins expression and the
279
decrease of cyclin A expression. This phenomenon is similar to those in other studies,
280
reporting that retigeric acid B, quinacrine and evodiamine induced cancer cell cycle
15
281
arrest in S phase (Liu, Liu, Xu, Young, Yuan, & Lou, 2010; Preet et al., 2012; Zhang,
282
Fan, Xu, Yang, Wang, & Liang, 2010).
283
284
3.5. Apoptosis
285
Since cell apoptosis may be one of the consequences of cell-cycle arrest, we
286
examined whether PAH2.5 induced apoptosis in MCF-7 cells. We stained the cells
287
with FITC Annexin V and PI, and we conducted internucleosomal DNA
288
fragmentation assays. Fig. 5A shows that after the treatment of PAH2.5 for 72 h with
289
various concentrations (0, 0.5, 1 and 1.5 mg/mL), the percentages of apoptotic cells
290
were 220, 277 and 349 % for MCF-7 cell lines as compared to the control with the
291
baseline of 100% (Fig. 5B). These results indicated that PAH2.5 induced apoptosis of
292
MCF-7 cell lines in a concentration-dependent manner.
293
The expression of apoptosis-related proteins was investigated in order to analyze
294
the underlying mechanisms by Western blot analysis. As shown in Fig. 6, PAH2.5
295
downregulated the expression of Bcl-2, PARP and caspase 9, and upregulated the
296
expression of p53, Bax and cleaved caspase 3 at the concentrations of 0.5 and 1
297
mg/mL. These results suggested that PAH2.5 induced apoptosis of MCF-7 cells by
298
activating caspase-related proteins family and might be through mitochondria
299
mediated pathway. This is in agreement with a previous study of activating p53 (Sax,
16
300
Fei, Murphy, Bernhard, Korsmeyer, & El-Deiry, 2002; Vogelstein, Lane, & Levine,
301
2000; Yamaguchi, Chen, Bhalla, & Wang, 2004) and triggering relocalisation of
302
cytochrome c lead to apoptosis of cancer cells (Pan, Becker, & Gerhauser, 2005;
303
Szigeti et al., 2010).
304
305
3.6. Amino acid sequences of peptides in PAH2.5
306
PAH2.5 was used to identify the amino acid sequences of peptides by MALDI
307
TOF/TOF MS/MS. Two peptides with the molecular mass of 2449.292 and 2562.405
308
were determined. After the analysis by MS/MS spectra processing with BioTools
309
database,
310
KPEGMDPPLSEPEDRRDGAAGPK
311
respectively (Fig. S2). The both peptides are rich in hydrophobic amino acids, such as
312
Pro, Leu, Ala and Phe. A previous study revealed that peptide with greater
313
hydrophobicity showed strong anticancer activity against cancer cell lines, including
314
MCF-7 (Huang et al., 2011). Therefore, PAH2.5 showed great antiproliferative activity
315
of MCF-7 probably attributed to the high hydrophobicity of the peptides.
the
amino
acid
sequences
and
of
the
two
peptides
are
KLPPLLLAKLLMSGKLLAEPCTGR,
316
317
318
4. Conclusions
PAH2.5 showed antiproliferative effect, induced cell cycle arrest in S phase and
17
319
apoptosis against MCF-7 cells. Two peptides in PAH2.5 with the molecular mass of
320
2449.292 and 2562.405 and their amino acid sequences were also identified. This
321
study has clearly demonstrated that tuna cooking juice has the potential to be a valid
322
protein source of bioactive peptides with the antiproliferative effect on MCF-7 cells
323
and without affecting normal breast epithelial cells.
324
325
Acknowledgments
326
This study was financially supported by National Science Council, Taiwan, Republic
327
of China, with the grant No. NSC 99-2313-B-039-001-MY3.
328
18
329
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Figure Legends
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Fig. 1.
451
Effect of protein hydrolysates derived from tuna cooking juice on cell proliferation of
452
MCF-7 cells cultured for 72 h in cell culture medium containing 1 mg/mL of
453
hydrolysates. Bars represent standard deviations from triplicate determinations.
454
Different letters indicate the significant differences (P < 0.05)
(A) Degree of hydrolysis (DH) of tuna cooking juice during hydrolysis. (B)
455
456
Fig. 2.
457
h at the concentration of 1 mg/mL. (B) Cell proliferation of MCF-7 treated with
458
PAH2.5 for 72 h at various concentrations. Bars represent standard deviations from
459
triplicate determinations.
(A) Cell proliferation of MCF-7 treated with the UF fractions of PAH for 72
460
461
Fig. 3.
462
treatment of PAH2.5 at various concentrations for 72 h, MCF-7 cells were harvested,
463
stained with propidium iodide, and analysed by flow cytometry. Flow cytometric
464
histograms are representative of three separate experiments. (B) Quantification of
465
percentage of MCF-7 cells treated by PAH2.5 in cell cycle. Bars represent standard
466
deviations from triplicate determinations. Different letters indicate the significant
467
differences (P < 0.05)
Effect of PAH2.5 on the cell cycle progression of MCF-7. (A) After the
26
468
469
Fig. 4.
470
investigated by Western blot analysis. Cells were treated with 0.5, 1 and 1.5 mg/mL
471
of PAH2.5 for 72 h. β-actin was used as a loading control. Figures showed the
472
representative blots from one of three experiments that gave similar results.
Effect of PAH2.5 on cell cycle-related protein levels in MCF-7 cells
473
474
Fig. 5.
475
of PAH2.5. (A) Flow cytometric analysis of PS externalization (annexin V binding)
476
and cell membrane integrity (PI staining). Cells were treated with 0, 0.5, 1 and 1.5
477
mg/mL of PAH2.5 for 72 h. The dual parameter dot plots combining annexin V-FITC
478
and PI fluorescence show the vial cell population in the lower left quadrant (Q3), the
479
early apoptotic cells in the lower right quadrant (Q4), and the late apoptotic cells in
480
the upper right quadrant (Q2). (B) Percentages of apoptotic cells (Q2 + Q4). Bars
481
represent standard deviations from triplicate determinations. Different letters indicate
482
the significant differences (P < 0.05).
Apoptosis assessment of MCF-7 cells treated with indicated concentrations
483
484
Fig. 6.
485
investigated by Western blot analysis. Cells were treated with 0.5, 1 and 1.5 mg/mL
486
PAH2.5 for 72 h. β-actin was used as a loading control. Figures show the
Effect of PAH2.5 on apoptosis-related protein levels in MCF-7 cells
27
487
representative blots from one of three experiments that gave similar results.
488
489
Fig. S1. Cell proliferation of MCF-10A treated with PAH2.5 for 72 h at various
490
concentrations. Bars represent standard deviations from triplicate determinations.
491
492
Fig. S2. MALDI-TOF/TOF MS/MS spectrum of the peptides in PAH2.5.
493
494
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