research article roe protein hydrolysates of giant grouper...

Research ArticleRoe Protein Hydrolysates of Giant Grouper(Epinephelus lanceolatus) Inhibit Cell Proliferation of OralCancer Cells Involving Apoptosis and Oxidative Stress

Jing-Iong Yang1 Jen-Yang Tang234 Ya-Sin Liu1 Hui-Ru Wang5 Sheng-Yang Lee67

Ching-Yu Yen68 and Hsueh-Wei Chang591011

1 Department of Seafood Science National Kaohsiung Marine University Kaohsiung 81157 Taiwan2 Department of Radiation Oncology Faculty of Medicine College of Medicine Kaohsiung Medical UniversityKaohsiung 80708 Taiwan

3 Department of Radiation Oncology Kaohsiung Medical University Hospital Kaohsiung 80708 Taiwan4 Department of Radiation Oncology Kaohsiung Municipal Ta-Tung Hospital Kaohsiung 80708 Taiwan5 Department of Biomedical Science and Environmental Biology Kaohsiung Medical University Kaohsiung 80708 Taiwan6 School of Dentistry Taipei Medical University Taipei 11031 Taiwan7 Division of Orthodontics Wan-Fang Medical Center Taipei Medical University Taipei 11648 Taiwan8 Department of Oral and Maxillofacial Surgery Chi-Mei Medical Center Tainan 71004 Taiwan9 Institute of Medical Science and Technology National Sun Yat-sen University Kaohsiung 80424 Taiwan10Center for Research Resources and Development of Kaohsiung Medical University Kaohsiung 80708 Taiwan11Cancer Center Kaohsiung Medical University Hospital Kaohsiung Medical University Kaohsiung 80708 Taiwan

Correspondence should be addressed to Ching-Yu Yen ycysmcgmailcom and Hsueh-Wei Chang changhwkmuedutw

Received 30 December 2015 Revised 1 March 2016 Accepted 21 March 2016

Academic Editor Blanca Hernandez-Ledesma

Copyright copy 2016 Jing-Iong Yang et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Roe protein hydrolysateswere reported to have antioxidant property but the anticancer effectswere less addressed especially for oralcancer In this study we firstly used the ultrafiltrated roe hydrolysates (URH) derived fromgiant grouper (Epinephelus lanceolatus) toevaluate the impact of URHon proliferation against oral cancer cellsWe found that URHdose-responsively reduced cell viability oftwo oral cancer cells (Ca9-22 andCAL 27) in terms of ATP assay Using flow cytometry URH-induced apoptosis of Ca9-22 cells wasvalidated by morphological features of apoptosis sub-G1 accumulation and annexin V staining in dose-responsive manners URHalso induced oxidative stress in Ca9-22 cells in terms of reactive oxygen species (ROS)superoxide generations and mitochondrialdepolarization Taken together these data suggest that URH is a potential natural product for antioral cancer therapy

1 Introduction

Oral cancer is the sixth most common cancer in the world[1 2] Although some oral tumormarkers had been identifiedfor detection [3] the oral cancer riskwas unable to be reduceddue to its nontherapeutic function Oral carcinogenesis isa complex and long-term multifocal process [4] Thereforethe drugs with chemoprevention are still needed for antioralcancer therapy

Fish protein hydrolysates from cobia (Rachycentroncanadum) [5 6] tilapia (Oreochromis spp) [7] grass carp(Ctenopharyngodon idellus) [8] fresh water carp (Catla catla)[9] and other species [10] exhibited the antioxidant prop-erty Furthermore the roe protein hydrolysates from defat-ted skipjack (Katsuwonous pelamis) [11] Channa striatusLabeo rohita [12] Cyprinus carpio and Epinephelus tauvina[13] had been found to possess the antioxidant propertyThese antioxidant properties may have the health promoting

Hindawi Publishing CorporationBioMed Research InternationalVolume 2016 Article ID 8305073 12 pageshttpdxdoiorg10115520168305073

2 BioMed Research International

effects such as anti-inflammation and antibacterial [14 15]However the anticancer effect of roe protein hydrolysates-derived antioxidants may have different properties and is lessaddressed

Drugs and natural products with the antioxidant effectsalso reported to inhibit cancer cell proliferation For examplethe grape seed extracts [16] red algal methanol extract [1718] and red algal ethanol extract [19] had been reportedto be antiproliferative to oral cancer cells Accordingly thepossible antiproliferative effect of roe protein hydrolysates iswarranted for further investigation

Recently the protein hydrolysates of fish [20 21] marine[22 23] and plant [24] sources have been applied to cancertherapy study For example fish protein hydrolysates werefound to inhibit proliferation of human breast cancer (MCF-76 and MDA-MB-231) cells [20] Fractions from loachprotein hydrolysates prepared by papain digestion have beenreported to have the antioxidant and antiproliferative activ-ities against colon (Caco-2) cancer cells [21] Antioxidantand antiproliferative activities also have been reported inprotein hydrolysate of blood clam (Tegillarca granosa) muscleagainst prostate lung and cervical cancer cells [22] bioactivepeptides from enzymatic hydrolysate of oyster (Saccostreacucullata) against colon cancer cells [23] and bioactivepeptides from chickpea (Cicer arietinum L) against breastcancer cells [24] However the performance of these proteinhydrolysates in oral cancer cells remains unclear

Giant grouper is the largest specie of groupers in TaiwanThe diameter of a fresh roe is from 2 to 3mm Due to its fastgrowth and high price giant grouper currently is regarded asa favorite species for marine culture in Taiwan [25] Howeverduring the massive seed production a large number of roeshave been collected because they failed to hatch To make theuse of the protein byproduct the enzymatic hydrolysis can beimplemented to enhance the bioactivities of the roe proteinhydrolysates

Therefore the subject of this study is to examine thepossible antiproliferative function of fish roe hydrolysatesof giant grouper (Epinephelus lanceolatus) against oral can-cer cells and explore its detailed mechanisms in terms ofcell viability cell cycle analysis apoptosis reactive oxygenspecies (ROS)superoxide generations and mitochondrialmembrane potential

2 Materials and Methods

21 Preparation of Defatted Roe Fresh fish roes of giantgroupers (E lanceolatus) were obtained from farm pondsin Pingtung Taiwan during July 2013 The samples wereplaced in ice and transported to the Department of SeafoodScience Kaohsiung Marine University within 1 h The wholeroes were cleaned using cool water (4∘C) and homogenizedin a cool room The homogenized roes were then freeze-dried Afterward lipids of the dried roe powders wereextracted as described previously [26] In brief each 100 gfreeze-dried homogenized roes were added to 300mL hexanefor 2 h fat extraction This procedure was repeated threetimes Moreover the solvents were evaporated by vacuumconcentration The defatted roe protein samples were then

freeze-dried The defatted roe powders were kept in sealedpolyethylene bag and then placed at minus40∘C until use

22 Preparation of Roe Protein Hydrolysate Roe proteinhydrolysate (RPH) was prepared from defatted grouperroe powder using Protease N (Amano Pharmaceutical CoNagoya Japan)The defatted sample (5 g drymatter) was sus-pended in a 250mL of pH 8 phosphate buffer The hydrolysisreaction was initiated by the ratio of Protease Nroe sampleat 1 100 (ww solid matter) The reaction was conducted atpH 8 and 50∘C for 9 h The enzymatic hydrolysis was endedby heating the mixtures at 90∘C for 10 min to inactivate theprotease activity The solution containing hydrolysate wascentrifuged at 5000 g for 10min at 4∘C (05PR-22 centrifugeHitachi Tokyo Japan) Then the supernatants were desaltedand lyophilized to dried RPH for storage at minus40∘C

23 Preparation of Ultrafiltrates from RPH through Centrifu-gal Ultrafiltration Filters The lyophilized RPH (obtainedfrom 9 h Protease N hydrolysis) was subsequently dissolvedin deionized distilled water The solution containing RHPwas processed through centrifugal ultrafiltration (UF) filters(Millipore Bedford MA USA) as described previously [5]RPH solution (12mL) was first passed through a centrifu-gal filter with 10 kDa MWCO and then its permeate waspassed through the UF membranes with 5 kDa MWCOThe permeates (ultrafiltrated roe hydrolysates URH) withmolecular size below 5 kDa were obtained The URH filtratewas lyophilized and stored at minus40∘C until use

24 Amino Acid Analysis of URH TheURHwere hydrolyzedwith 6NHCl at 110∘C for 24 h under vacuumThe amino acidanalysis was performed using the Pico-Tag system (WatersMilford MA) as described [27]

25 Cell Cultures Two human oral cancer cell lines (Ca9-22and CAL 27) were available from the Japanese Collection ofResearch Bioresources (JCRB) Cell Bank (National Instituteof Biomedical Innovation Osaka Japan) and the AmericanType Culture Collection (ATCC Virginia USA) respectively[28] Cells were maintained in DMEMF12 (3 2) medium(Gibco Grand Island NY USA) supplemented with 10fetal bovine serum (Gibco) 100UmL penicillin 100 120583gmLstreptomycin and 003 glutamine under the humidifiedincubator at 37∘C with 5 CO

2

26 Cell Viability URH was dissolved in culture mediumfor cell treatment Cells were plated at 4000 cellswell in96-well plates After plating overnight cells were treatedwith URH at indicated concentrations (0 025 05 0751 15 2 and 25mgmL) for 24 h Then cellular ATP levelwas analyzed by the ATP-lite Luminescence ATP DetectionAssay System (PerkinElmer Life Sciences Boston MA USA)according to themanufacturerrsquos instructions [29] Finally theluminescence was detected using a microplate luminometer(CentroPRO LB 962 Berthold ND USA)

27 Cell Cycle Distribution Cellular DNA was detected bypropidium iodide (PI) (Sigma St Louis MO USA) [30]

BioMed Research International 3

In brief 3 times 105 cells per well in 6-well plates were platedovernight Cells were treated with 0 05 1 15 2 and25mgmL of URH for 24 h After harvest cells were washedwith PBS and fixed overnight with 70 ethanol Finally thecells were resuspended in 50120583gmL PI in PBS for 30minat 37∘C in darkness Cell cycle distribution was determinedby a flow cytometer (BD Accuri C6 Becton-DickinsonMansfield MA USA) and a BD Accuri C6 Software (version10264)

28 Apoptosis by Annexin VPI The apoptosis-like (sub-G1)status was further examined by annexin V (Strong BiotectCorporation Taipei Taiwan)PI as described [31] In brief3times10

5 cells per well in 6-well plates were plated for 24 h Cellswere treated with the indicated concentrations of URH for24 h After drug treatment cells were incubated with 100120583Lbinding buffer containing 2 120583L of annexin-V-fluoresceinisothiocyanate (FITC) stock (025 120583g120583L) and 2120583L of PI stock(1mgmL) for 30min Finally it was suspended with 400 120583LPBS for analysis by a flow cytometer (BD Accuri C6) and itssoftware

29 Intracellular ROS The fluorescent dye 2101584071015840-dichlorod-ihydrofluorescein diacetate (DCFH-DA) was used to detectROS [19] 3 times 105 cells per well in 6-well plates in 2mLmediumwere plated for 24 h Cells were treatedwith differentconcentrations of URH for 6 h After PBS washing 100 nMDCFH-DA in PBS was added to cells in 6-well platesunder an incubator for 30min After harvest PBS washingand centrifugation cells were resuspended in 1mL PBS foranalysis by a flow cytometer (BD Accuri C6) and its software

210 Intracellular Superoxide MitoSOXRedmitochondrialsuperoxide indicator (Molecular Probes Invitrogen EugeneOR USA) was reported to be the fluorescent dye for mito-chondrial superoxide [32] Assessing mitochondrial redoxstatus has been detected by flow cytometric methods [33]With a slight modification 3 times 105 cells per well in 6-well plates in 2mL medium were plated for 24 h Cellswere treated with different concentrations of URH for 1 hSubsequently 5 120583M MitoSOX was added to cells in 6-wellplates under an incubator for 10min After harvest PBSwashing and centrifugation cells were resuspended in 1mLPBS for analysis by a flow cytometer (BD Accuri C6) and itssoftware

211 Mitochondrial Membrane Potential (MMP) MitoP-robe DiOC

2(3) assay kit (Invitrogen Eugene OR USA)

was used to measure MMP as described previously [34]Briefly 3 times 105 cells in 2mL medium per well in 6-well platewere plated and incubated for 24 h Cells were treated withURH treatment for 24 h Subsequently 50 nM DiOC

2(3) was

added per well under an incubator for 30min After harvestcells were resuspended in 1mL PBS for analysis by a flowcytometer (BD Accuri C6) and its software

212 Statistical Analysis The significance of differences wasevaluated by Studentrsquos t-test in SigmaPlot 10 software (SystatSoftware Inc San Jose CA USA) All data were compared

Ca9-22CAL 27

lowastlowast

lowast lowastlowast

lowastlowastlowastlowast


lowastlowastlowastlowastlowastlowast

lowastlowast

lowastlowast

lowastlowast

025 050 075 100 150 200 250000URH (mgmL)

0

20

40

60

80

100

120

ATP

cont

ent (

)

Figure 1 Cell viabilities of two URH-treated oral cancer cells Oralcancer (Ca9-22 andCAL 27) cells were treated with 0 05 075 1 152 and 25mgmL of URH for 24 h incubation The cell viability wasmeasured by the ATP assay Data means plusmn SDs (119899 = 6) lowast119901 lt 005lowastlowast

119901

lt 0001 against control

with controls lowast and lowastlowast respectively indicate 119901 lt 005 and119901 lt 0001 against control

3 Results

31 Amino Acid Composition of URH As shown in Table 1the amino acid composition of URH indicates that URHwas composed of full kind of amino acids after purificationprocesses

32 Antiproliferation of URH With the cell viability ()in terms of ATP content measurement (Figure 1) two oralcancer cells (Ca9-22 and CAL 27) at indicated concentrationsof URH were dose-responsively decreased (119901 lt 005ndash0001compared to the control) The IC

50values of URH at 24 h

treatment for oral cancer Ca9-22 cells were 085mgmL andIC50value was undetectable for CAL 27 cells

33 Morphology Change of URH The cell morphology ofURH-treated oral cancer Ca9-22 cells was shown in Figure 2The morphological features of apoptosis including apoptoticbodies and shrinkage of the cells appeared at higher concen-tration of URH

34 Cell Cycle Disturbance of URH As shown in Figure 3(a)the flow cytometry-based cell cycle distribution patternsof URH-treated oral cancer Ca9-22 cells were displayedAfter URH treatment (Figure 3(b)) the sub-G1 () of URH-treated (0ndash25mgmL) Ca9-22 cells were dose-responsivelyincreased (119901 lt 0001) In contrast the G0G1 S and G2M() of URH-treated Ca9-22 cells were dose-responsivelydecreased (119901 lt 005ndash0001)


25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

Figure 2 Cell morphology of URH-treated oral cancer Ca9-22 cells Cells were treated with 0 05 075 1 15 2 and 25mgmL of URH for24 h incubation Cell images were captured at 100x magnification

Table 1 Amino acid compositionlowast of URH

Amino acid ()Asp + Asn 979Glu + Gln 1276Ser 711Gly 784His 171Arg 345Thr 555Ala 1129Pro 821Tyr 249Val 657Met 167Cys 031Ile 479Leu 758Phe 266Lys 621lowastData are the mean values of duplicate determinations expressed as mil-ligram of amino acid per 100mg of URH

35 Annexin VPI-Based Apoptosis of URH To furtherexamine the role of apoptosis the flow cytometry-basedannexin VPI patterns of URH-treated oral cancer Ca9-22cells were performed (Figure 4(a)) As shown in Figure 4(b)the annexin V-positive intensities () for URH-treated (0ndash25mgmL) Ca9-22 cells were dose-responsively increased(119901 lt 005ndash0001)

36 ROSGeneration ofURH Since some apoptosis-inducibledrugs were associated with ROS generation [35ndash38] the roleof oxidative stress inURH-treated Ca9-22 cells was examinedin terms of ROS detection As shown in Figure 5(a) theflow cytometry-based ROS staining patterns of URH-treatedCa9-22 cells at 6 h incubation were displayed As shown inFigure 5(b) the relative ROS-positive intensities () ofURH-treated (0ndash2mgmL) Ca9-22 cells were dose-responsivelyinduced (119901 lt 005ndash0001)

37 Superoxide Generation of URH The role of oxidativestress in URH-treated Ca9-22 cells was examined in termsof superoxide detection As shown in Figure 6(a) the flowcytometry-based superoxide staining (MitoSOX) patternsof URH-treated (0ndash25mgmL) Ca9-22 cells at 1 h incu-bation were displayed As shown in Figure 6(b) the rela-tive MitoSOX-positive intensities () of URH-treated (0ndash25mgmL) Ca9-22 cells were dose-responsively induced(119901 lt 005ndash0001)

38 MMP of URH The role of oxidative stress in URH-treated Ca9-22 cells was also examined in terms of MMP byflow cytometry As shown in Figure 7(a) the MMP stainingpatterns of URH-treated Ca9-22 cells at 24 h incubationwere displayed As shown in Figure 7(b) the MMP-positiveintensities () of URH-treated (0ndash25mgmL) Ca9-22 cellswere dose-responsively decreased (119901 lt 0001) (Figure 7(b))

4 Discussion

Fish protein hydrolysates were well-known for the antiox-idant property Although most studies of fish protein


Sub-G1G2M

S

G1

Sub-G1 S Sub-G1G2M

S

G1

Sub-G1 S Sub-G1G2M

G2M

G2M G2M

S

G1

G1 G1

G1

Sub-G1 S

25mgmL

times105

times105times10

5

times105

times105

times105

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

(a)

5846 plusmn 097lowastlowast






1449 plusmn 125lowast







4752 plusmn 059




4827 plusmn 204



327 plusmn 005

4807 plusmn 078

1755 plusmn 075

3111 plusmn 095

252151050URH (mgmL)

Sub-G1 ()G1 ()S ()G2M ()

(b)

Figure 3 The cell cycle changes of URH-treated oral cancer Ca9-22 cells Cells were treated with 0 05 1 15 2 and 25mgmL of URH for24 h (a) Representative cell cycle distribution patterns of flow cytometry of URH-treated Ca9-22 cells The cell cycle phases were labeled ineach panel (b) Quantification analysis for the cell cycle phases in Figure 3(a) Data mean plusmn SD (119899 = 3) lowast119901 lt 005 and lowastlowast119901 lt 0001 againstcontrol

hydrolysates had been reported the possible anticancereffect was less addressed In current study we chose theEpinephelus lanceolatus-derived roe hydrolysates (URH) andvalidated their antiproliferative effect against oral cancercells We found that URH induced antiproliferation sub-G1accumulation apoptosis ROS generation andmitochondrialdepolarization of oral cancer cells

In the current study the fish roe protein hydrolysates-derivedURH is the ultrafiltration fractionwith lowmolecularweights (MW) (lt5 kDa) and it showed that the IC

50values at

24 h treatment for oral cancer Ca9-22 cells were 085mgmLin terms of ATP assayThe IC

30(70 viability) values of URH

at 24 h treatment for oral cancer Ca9-22 and CAL 27 cellswere 05 and 15mgmL respectively Similarly the muscletissue-derived loach (Misgurnus anguillicaudatus) protein

hydrolysates (LPH) prepared by papain digestion displayeddifferential antiproliferative activities against breast colonand liver cancer cells for different ultrafiltration fractionswith different MW ranges [21] Based on MTS assay the IC

50

values of LPH-III (MW ranging from 3 to 5 kDa) at 96 htreatment were 33 15 and 22mgmL for breast (MCF-7)colon (Caco-2) and liver (HepG2) cancer cells respectivelyIC50

values of LPH-IV (MW ranging from lt3 kDa) at 96 htreatment were 16 10 and 13mgmL for MCF-7 Caco-2and HepG2 cancer cells respectively Accordingly the LPHfractions with the low MW ranging from lt5 kDa (LPH-IIIand LPH-IV) have detectable IC

50values at 96 h treatment

In contrast the IC50

values of the LPH fractions with thehigh MW (5ndash10 and gt10 kDa) were undetectable Thereforeantiproliferative activities against cancer cells may be varied


105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI10

3

104

105

106

PI

105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

0

5

10

15

20

Apop

totic

cells

()

(b)

Figure 4 AnnexinVPI-based apoptosis ofURH-treated oral cancerCa9-22 cells Ca9-22 cells were treatedwith 0 05 1 15 2 and 25mgmLof URH for 24 h (a) Representative results of flow cytometry-based annexin VPI double staining of URH-treated Ca9-22 cells Annexin V(+)PI (+) and annexin V (+)PI (minus) were calculated as the apoptosis (+) in each panel (b) Quantification analysis of apoptosis for URH-treated Ca9-22 cells in Figure 4(a) Data mean plusmn SD (119899 = 3) lowastlowast119901 lt 0001 against control


(+) (+) (+)

(+) (+)

V1-L V1-R V1-L V1-R V1-L V1-R

V1-L V1-R V1-L V1-R

500 500 235 765 289 711

319 681 200 800

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000C

ount

0

500

1000

Cou

nt

1mgmL

15mgmL 2mgmL

0mgmL 05mgmL

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity10

310

510

710

1

ROS intensity

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowast

05 10 15 2000URH (mgmL)

80

100

120

140

160

180

Relat

ive R

OS

(+) (

)

(b)

Figure 5 ROS generation of URH-treated oral cancer Ca9-22 cells Cells were treated with 0 05 1 15 and 2mgmL of URH for 6 h (a)Representative ROS patterns of flow cytometry for URH-treated Ca9-22 cells In each panel the right side labeled with (+) indicates theROS-positive region (b) Quantification analysis of relative ROS intensity in Figure 5(a) Data mean plusmn SD (119899 = 3) lowast119901 lt 005 and lowastlowast119901 lt 0001against control


(+) (+) (+)

(+) (+) (+)

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

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Cou

nt

0

400

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Cou

nt

0

400

800

Cou

nt



938 62 944 56 900 100

881 119878 122 849 152

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

(a)

lowastlowast


lowastlowast

05 10 15 20 2500URH (mgmL)

80

100

120

140

160

180

200

220

240

260

280

Relat

ive m

itoSO

X (+

) (

)

(b)

Figure 6 Superoxide generation of URH-treated oral cancer Ca9-22 cells (a) Ca9-22 cells treated with 0 05 1 15 2 and 25mgmL ofURH for 1 h were stained with MitoSOX dye The right side labeled with (+) indicates the MitoSOX-positive region in each panel (b)Quantification analysis of relative MitoSOX (+) fluorescent intensity () Data mean plusmn SD (119899 = 3) lowastlowast119901 lt 0001 against control


(+) (+) (+)

(+) (+) (+)

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

80

84

88

92

96

100

MM

P (+

) (

)

(b)

Figure 7 MMP change of URH-treated oral cancer Ca9-22 cells Cells were treated with 0 05 1 15 2 and 25mgmL of URH for 24 h (a)Representative MMP patterns of flow cytometry for URH-treated Ca9-22 cells In each panel the horizontal line labeled with (+) in theright side indicates the MMP-positive region (b) Quantification analysis of MMP intensity ((+)) in Figure 7(a) Data mean plusmn SD (119899 = 3)lowastlowast

119901



with different ultrafiltration fractions with different MWranges Low MW protein hydrolysates seem to be morepotential to anticancer cell proliferation

The antiproliferative effect of several hydrolysates fromdifferent parts of the marine species had been reported inother cancer types such as breast [20] colon and liver [39]cancers For example peptide-rich fish hydrolysates derivedfrom fresh filleting by-products or headed and gutted by-catches of blue whiting exerted a significant antiproliferativeactivity at 1mgmL for 72 h treatment with growth inhibitionof 223ndash263 on breast cancer MCF-76 cells and 135ndash298 on breast cancer MDA-MB-231 cells using MTS assay[20] Because these protein hydrolysate studies mentionedabove were derived from different parts and species of fishesand they were treated with different incubation times itwas suitable to compare their drug sensitivity between eachother In general our experiment was performed in shortertreatment time and displayed the lower IC

50values of fish

protein hydrolysates (URH) in inhibiting proliferation of oralcancer cells It is also possible that drug sensitivity of fishprotein hydrolysates in anticancer cell effect may be cancercell-type dependent Moreover it was warranted for furtherinvestigation that the fish protein hydrolysates from roe andother parts may have different potentials for antiproliferationof oral cancer cells

Protein hydrolysates from many species were reported tohave the antioxidant property [5ndash13]The dual roles of antiox-idants can explain why protein hydrolysates with antioxidantproperty also display anticancer effect [40] Oxidative stresshad been reviewed to regulate the endoplasmic reticulumstress [41] autophagy [42] and apoptosis [43] leading tocell death For exogenous antioxidants it may behave likethe double-edged swords in cellular redox state that is itis protective at physiologic doses but it is harmful at highdoses [40] In current study we provided evidence for theapoptosis effect of URH in oral cancer Ca9-22 cells suchas sub-G1 accumulation and annexin VPI staining (Figures3 and 4) which were coupled with high ROS superoxidegenerations and mitochondrial depolarization that is thecorrelation values (1198772) are 08435 06294 and 06782 for ROS(+) mitoSOX (+) and MMP (minus) versus apoptosis (annexinV (+)) respectively although the effect of lower doses ofURHwas not examined in our study Similarly the grape seedextract (GSE) displayed the normal proliferation at low dosesbut demonstrated the antiproliferation for oral cancer cells athigh doses [16] Moreover GSE at high doses displayed highROS generation andmitochondrial depolarization than thoseof the low doses [16] Therefore the differential oxidativestress may partly contribute the dual roles of antioxidants

5 Conclusion

URH is the ultrafiltration fraction of fish roe proteinhydrolysates with low MW In current study we firstlydemonstrated that URH can inhibit cell proliferation of twooral cancer cells (Ca9-22 and CAL 27) URH also induced thecharacters of apoptosis of oral cancer cells such as apoptoticmorphology change sub-G1 accumulation and annexinVPI positive expression This antiproliferative mechanism

includes the ROS and superoxide generations and mito-chondrial depolarizationTherefore these results suggest thatURH has an apoptosis-based anticancer potential for oralcancer therapy

Competing Interests

The authors declare no competing interests

Acknowledgments

This work was supported by funds of the Ministry ofScience and Technology (MOST 104-2320-B-037-013-MY3)the National Kaohsiung Marine University Research Project(NKMU-9910286) Kaohsiung Medical University ldquoAim forthe Top Universities Grant no KMU-TP104PR02rdquo theNational SunYat-senUniversity-KMU Joint Research Project(no NSYSU-KMU 105-p002) the Kaohsiung Municipal Ta-Tung Hospital (kmtth-104-003) the Health and WelfareSurcharge of Tobacco Products the Ministry of Health andWelfare Taiwan Republic of China (MOHW105-TDU-B-212-134005) and ChiMei-KMU Joint Project (104CM-KMU-02)

References

[1] S Warnakulasuriya ldquoGlobal epidemiology of oral and oropha-ryngeal cancerrdquo Oral Oncology vol 45 no 4-5 pp 309ndash3162009

[2] P E Petersen ldquoOral cancer prevention and controlmdashtheapproach of the World Health Organizationrdquo Oral Oncologyvol 45 no 4-5 pp 454ndash460 2009

[3] C-Y Yen C-Y HuangM-F Hou et al ldquoEvaluating the perfor-mance of fibronectin 1 (FN1) integrin12057241205731 (ITGA4) syndecan-2 (SDC2) and glycoprotein CD44 as the potential biomarkersof oral squamous cell carcinoma (OSCC)rdquo Biomarkers vol 18no 1 pp 63ndash72 2013

[4] T Tanaka M Tanaka and T Tanaka ldquoOral carcinogenesisand oral cancer chemoprevention a reviewrdquo Pathology ResearchInternational vol 2011 Article ID 431246 10 pages 2011

[5] J-I Yang H-Y Ho Y-J Chu and C-J Chow ldquoCharacteristicand antioxidant activity of retorted gelatin hydrolysates fromcobia (Rachycentron canadum) skinrdquo Food Chemistry vol 110no 1 pp 128ndash136 2008

[6] J-I Yang W-S Liang C-J Chow and K J Siebert ldquoProcessfor the production of tilapia retorted skin gelatin hydrolysateswith optimized antioxidative propertiesrdquo Process Biochemistryvol 44 no 10 pp 1152ndash1157 2009

[7] C-J Chow and J-I Yang ldquoThe effect of process variablesfor production of cobia (Rachycentron canadum) skin gelatinhydrolysates with antioxidant propertiesrdquo Journal of Food Bio-chemistry vol 35 no 3 pp 715ndash734 2008

[8] X Li Y Luo H Shen and J You ldquoAntioxidant activitiesand functional properties of grass carp (Ctenopharyngodonidellus) protein hydrolysatesrdquo Journal of the Science of Food andAgriculture vol 92 no 2 pp 292ndash298 2012

[9] K Elavarasan V Naveen Kumar and B A Shamasun-dar ldquoAntioxidant and functional properties of fish proteinhydrolysates from fresh water carp (Catla catla) as influenced


by the nature of enzymerdquo Journal of Food Processing andPreservation vol 38 no 3 pp 1207ndash1214 2014

[10] M Chalamaiah B Dinesh Kumar R Hemalatha and TJyothirmayi ldquoFish protein hydrolysates proximate composi-tion amino acid composition antioxidant activities and appli-cations a reviewrdquo FoodChemistry vol 135 no 4 pp 3020ndash30382012

[11] K Balaswamy P G Prabhakara Rao G Narsing Rao and TJyothirmayi ldquoFunctional properties of roe protein hydrolysatesfrom Catla catlardquo Electronic Journal of Environmental Agricul-tural and Food Chemistry vol 10 pp 2139ndash2147 2011

[12] N R Galla P R Pamidighantam S Akula and B KarakalaldquoFunctional properties and in vitro antioxidant activity of roeprotein hydrolysates of Channa striatus and Labeo rohitardquo FoodChemistry vol 135 no 3 pp 1479ndash1484 2012

[13] G N Rao ldquoPhysico-chemical functional and antioxidantproperties of roe protein concentrates from Cyprinus carpioand Epinephelus tauvinardquo Journal of Food and PharmaceuticalSciences vol 2 no 1 pp 15ndash22 2014

[14] J-C Lee M-F Hou H-W Huang et al ldquoMarine algal natu-ral products with anti-oxidative anti-inflammatory and anti-cancer propertiesrdquo Cancer Cell International vol 13 article 552013

[15] L Najafian andA S Babji ldquoA review of fish-derived antioxidantand antimicrobial peptides their production assessment andapplicationsrdquo Peptides vol 33 no 1 pp 178ndash185 2012

[16] C-Y Yen M-F Hou Z-W Yang et al ldquoConcentration effectsof grape seed extracts in anti-oral cancer cells involving dif-ferential apoptosis oxidative stress and DNA damagerdquo BMCComplementary and Alternative Medicine vol 15 article 942015

[17] C-C Yeh C-N Tseng J-I Yang et al ldquoAntiproliferation andinduction of apoptosis in Ca9-22 oral cancer cells by ethanolicextract of Gracilaria tenuistipitatardquoMolecules vol 17 no 9 pp10916ndash10927 2012

[18] Y-H Yen A A Farooqi K-T Li et al ldquoMethanolic extracts ofSolieria robusta inhibits proliferation of oral cancer Ca9-22 cellsvia apoptosis and oxidative stressrdquoMolecules vol 19 no 11 pp18721ndash18732 2014

[19] C-C Yeh J-I Yang J-C Lee et al ldquoAnti-proliferative effect ofmethanolic extract of Gracilaria tenuistipitata on oral cancercells involves apoptosis DNA damage and oxidative stressrdquoBMC Complementary and Alternative Medicine vol 12 article142 2012

[20] L Picot S Bordenave SDidelot et al ldquoAntiproliferative activityof fish protein hydrolysates on human breast cancer cell linesrdquoProcess Biochemistry vol 41 no 5 pp 1217ndash1222 2006

[21] L You M Zhao R H Liu and J M Regenstein ldquoAntioxidantand antiproliferative activities of loach (Misgurnus anguilli-caudatus) peptides prepared by papain digestionrdquo Journal ofAgricultural and Food Chemistry vol 59 no 14 pp 7948ndash79532011

[22] C-F Chi F-Y Hu B Wang T Li and G-F Ding ldquoAntioxidantand anticancer peptides from the protein hydrolysate of bloodclam (Tegillarca granosa) musclerdquo Journal of Functional Foodsvol 15 pp 301ndash313 2015

[23] S Umayaparvathi S Meenakshi V Vimalraj M ArumugamG Sivagami and T Balasubramanian ldquoAntioxidant activityand anticancer effect of bioactive peptide from enzymatichydrolysate of oyster (Saccostrea cucullata)rdquo Biomedicine ampPreventive Nutrition vol 4 no 3 pp 343ndash353 2014

[24] Z H Xue H C Wen L J Y Zhai et al ldquoAntioxidant activityand anti-proliferative effect of a bioactive peptide from chickpea(Cicer arietinum L)rdquo Food Research International vol 77 pp75ndash81 2015

[25] J-R Hseu P-P Hwang and Y-Y Ting ldquoMorphometric modeland laboratory analysis of intracohort cannibalism in giantgrouper Epinephelus lanceolatus fryrdquo Fisheries Science vol 70no 3 pp 482ndash486 2004

[26] E G Bligh and W J Dyer ldquoA rapid method of total lipidextraction and purificationrdquo Canadian Journal of Biochemistryand Physiology vol 37 no 8 pp 911ndash917 1959

[27] K Sato Y Tsukamasa C Imai K Ohtsuki Y Shimizu andM Kawabata ldquoImproved method for identification and deter-mination of 120576-(120574-glutamyl)-lysine cross-link in protein usingproteolytic digestion and derivatization with phenyl isothio-cyanate followed by high performance liquid chromatographyseparationrdquo Journal of Agricultural and Food Chemistry vol 40pp 806ndash810 1992

[28] L Jiang N Ji Y Zhou et al ldquoCAL 27 is an oral adenosquamouscarcinoma cell linerdquo Oral Oncology vol 45 no 11 pp e204ndashe207 2009

[29] J Wei J L Stebbins S Kitada et al ldquoAn optically pureapogossypolone derivative as potent pan-active inhibitor ofanti-apoptotic Bcl-2 family proteinsrdquo Frontiers in Oncology vol1 article 28 2011

[30] B-H Chen H-W Chang H-M Huang et al ldquo(-)-Anonaineinduces DNA damage and inhibits growth and migration ofhuman lung carcinomaH1299 cellsrdquo Journal of Agricultural andFood Chemistry vol 59 no 6 pp 2284ndash2290 2011

[31] C-C Chiu P-L Liu K-J Huang et al ldquoGoniothalamininhibits growth of human lung cancer cells through DNAdamage apoptosis and reduced migration abilityrdquo Journal ofAgricultural and Food Chemistry vol 59 no 8 pp 4288ndash42932011

[32] P Mukhopadhyay M Rajesh K Yoshihiro G Hasko andP Pacher ldquoSimple quantitative detection of mitochondrialsuperoxide production in live cellsrdquoBiochemical andBiophysicalResearch Communications vol 358 no 1 pp 203ndash208 2007

[33] R Li N Jen F Yu and T K Hsiai ldquoAssessing mitochondrialredox status by flow cytometric methods vascular response tofluid shear stressrdquo Current Protocols in Cytometry chapter 9unit 937 2011

[34] C-Y Yen C-C Chiu R-W Haung et al ldquoAntiprolifera-tive effects of goniothalamin on Ca9-22 oral cancer cellsthrough apoptosis DNAdamage andROS inductionrdquoMutationResearchGenetic Toxicology and Environmental Mutagenesisvol 747 no 2 pp 253ndash258 2012

[35] C-Y Yen M-H Lin S-Y Liu et al ldquoArecoline-mediated inhi-bition ofAMP-activated protein kinase through reactive oxygenspecies is required for apoptosis inductionrdquo Oral Oncology vol47 no 5 pp 345ndash351 2011

[36] M H Han C Park C-Y Jin et al ldquoApoptosis induction ofhuman bladder cancer cells by sanguinarine through reac-tive oxygen species-mediated up-regulation of early growthresponse gene-1rdquo PLoS ONE vol 8 no 5 Article ID e634252013

[37] L Raj T Ide A U Gurkar et al ldquoSelective killing of cancercells by a small molecule targeting the stress response to ROSrdquoNature vol 475 no 7355 pp 231ndash234 2011


[38] H Ding C Han D Guo et al ldquoSelective induction of apoptosisof human oral cancer cell lines by avocado extracts via a ROS-mediated mechanismrdquo Nutrition and Cancer vol 61 no 3 pp348ndash356 2009

[39] A Kannan N S Hettiarachchy M Marshall S Raghavanand H Kristinsson ldquoShrimp shell peptide hydrolysates inhibithuman cancer cell proliferationrdquo Journal of the Science of Foodand Agriculture vol 91 no 10 pp 1920ndash1924 2011

[40] J Bouayed and T Bohn ldquoExogenous antioxidantsmdashdouble-edged swords in cellular redox state health beneficial effectsat physiologic doses versus deleterious effects at high dosesrdquoOxidativeMedicine and Cellular Longevity vol 3 no 4 pp 228ndash237 2010

[41] A A Farooqi K-T Li S Fayyaz et al ldquoAnticancer drugs forthe modulation of endoplasmic reticulum stress and oxidativestressrdquo Tumor Biology vol 36 no 8 pp 5743ndash5752 2015

[42] A A Farooqi S Fayyaz M-F Hou K-T Li J-Y Tang and H-W Chang ldquoReactive oxygen species and autophagymodulationin non-marine drugs and marine drugsrdquoMarine Drugs vol 12no 11 pp 5408ndash5424 2014

[43] J M Mates J A Segura F J Alonso and J MarquezldquoOxidative stress in apoptosis and cancer an updaterdquo Archivesof Toxicology vol 86 no 11 pp 1649ndash1665 2012

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


effects such as anti-inflammation and antibacterial [14 15]However the anticancer effect of roe protein hydrolysates-derived antioxidants may have different properties and is lessaddressed

Drugs and natural products with the antioxidant effectsalso reported to inhibit cancer cell proliferation For examplethe grape seed extracts [16] red algal methanol extract [1718] and red algal ethanol extract [19] had been reportedto be antiproliferative to oral cancer cells Accordingly thepossible antiproliferative effect of roe protein hydrolysates iswarranted for further investigation

Recently the protein hydrolysates of fish [20 21] marine[22 23] and plant [24] sources have been applied to cancertherapy study For example fish protein hydrolysates werefound to inhibit proliferation of human breast cancer (MCF-76 and MDA-MB-231) cells [20] Fractions from loachprotein hydrolysates prepared by papain digestion have beenreported to have the antioxidant and antiproliferative activ-ities against colon (Caco-2) cancer cells [21] Antioxidantand antiproliferative activities also have been reported inprotein hydrolysate of blood clam (Tegillarca granosa) muscleagainst prostate lung and cervical cancer cells [22] bioactivepeptides from enzymatic hydrolysate of oyster (Saccostreacucullata) against colon cancer cells [23] and bioactivepeptides from chickpea (Cicer arietinum L) against breastcancer cells [24] However the performance of these proteinhydrolysates in oral cancer cells remains unclear

Giant grouper is the largest specie of groupers in TaiwanThe diameter of a fresh roe is from 2 to 3mm Due to its fastgrowth and high price giant grouper currently is regarded asa favorite species for marine culture in Taiwan [25] Howeverduring the massive seed production a large number of roeshave been collected because they failed to hatch To make theuse of the protein byproduct the enzymatic hydrolysis can beimplemented to enhance the bioactivities of the roe proteinhydrolysates

Therefore the subject of this study is to examine thepossible antiproliferative function of fish roe hydrolysatesof giant grouper (Epinephelus lanceolatus) against oral can-cer cells and explore its detailed mechanisms in terms ofcell viability cell cycle analysis apoptosis reactive oxygenspecies (ROS)superoxide generations and mitochondrialmembrane potential

2 Materials and Methods

21 Preparation of Defatted Roe Fresh fish roes of giantgroupers (E lanceolatus) were obtained from farm pondsin Pingtung Taiwan during July 2013 The samples wereplaced in ice and transported to the Department of SeafoodScience Kaohsiung Marine University within 1 h The wholeroes were cleaned using cool water (4∘C) and homogenizedin a cool room The homogenized roes were then freeze-dried Afterward lipids of the dried roe powders wereextracted as described previously [26] In brief each 100 gfreeze-dried homogenized roes were added to 300mL hexanefor 2 h fat extraction This procedure was repeated threetimes Moreover the solvents were evaporated by vacuumconcentration The defatted roe protein samples were then

freeze-dried The defatted roe powders were kept in sealedpolyethylene bag and then placed at minus40∘C until use

22 Preparation of Roe Protein Hydrolysate Roe proteinhydrolysate (RPH) was prepared from defatted grouperroe powder using Protease N (Amano Pharmaceutical CoNagoya Japan)The defatted sample (5 g drymatter) was sus-pended in a 250mL of pH 8 phosphate buffer The hydrolysisreaction was initiated by the ratio of Protease Nroe sampleat 1 100 (ww solid matter) The reaction was conducted atpH 8 and 50∘C for 9 h The enzymatic hydrolysis was endedby heating the mixtures at 90∘C for 10 min to inactivate theprotease activity The solution containing hydrolysate wascentrifuged at 5000 g for 10min at 4∘C (05PR-22 centrifugeHitachi Tokyo Japan) Then the supernatants were desaltedand lyophilized to dried RPH for storage at minus40∘C

23 Preparation of Ultrafiltrates from RPH through Centrifu-gal Ultrafiltration Filters The lyophilized RPH (obtainedfrom 9 h Protease N hydrolysis) was subsequently dissolvedin deionized distilled water The solution containing RHPwas processed through centrifugal ultrafiltration (UF) filters(Millipore Bedford MA USA) as described previously [5]RPH solution (12mL) was first passed through a centrifu-gal filter with 10 kDa MWCO and then its permeate waspassed through the UF membranes with 5 kDa MWCOThe permeates (ultrafiltrated roe hydrolysates URH) withmolecular size below 5 kDa were obtained The URH filtratewas lyophilized and stored at minus40∘C until use

24 Amino Acid Analysis of URH TheURHwere hydrolyzedwith 6NHCl at 110∘C for 24 h under vacuumThe amino acidanalysis was performed using the Pico-Tag system (WatersMilford MA) as described [27]

25 Cell Cultures Two human oral cancer cell lines (Ca9-22and CAL 27) were available from the Japanese Collection ofResearch Bioresources (JCRB) Cell Bank (National Instituteof Biomedical Innovation Osaka Japan) and the AmericanType Culture Collection (ATCC Virginia USA) respectively[28] Cells were maintained in DMEMF12 (3 2) medium(Gibco Grand Island NY USA) supplemented with 10fetal bovine serum (Gibco) 100UmL penicillin 100 120583gmLstreptomycin and 003 glutamine under the humidifiedincubator at 37∘C with 5 CO

2

26 Cell Viability URH was dissolved in culture mediumfor cell treatment Cells were plated at 4000 cellswell in96-well plates After plating overnight cells were treatedwith URH at indicated concentrations (0 025 05 0751 15 2 and 25mgmL) for 24 h Then cellular ATP levelwas analyzed by the ATP-lite Luminescence ATP DetectionAssay System (PerkinElmer Life Sciences Boston MA USA)according to themanufacturerrsquos instructions [29] Finally theluminescence was detected using a microplate luminometer(CentroPRO LB 962 Berthold ND USA)

27 Cell Cycle Distribution Cellular DNA was detected bypropidium iodide (PI) (Sigma St Louis MO USA) [30]










2(3) was



Ca9-22CAL 27

lowastlowast

lowast lowastlowast




lowastlowast

lowastlowast

lowastlowast

025 050 075 100 150 200 250000URH (mgmL)

0

20

40

60

80

100

120

ATP

cont

ent (

)


119901



3 Results








25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL








4 Discussion



Sub-G1G2M

S

G1

Sub-G1 S Sub-G1G2M

S

G1

Sub-G1 S Sub-G1G2M

G2M

G2M G2M

S

G1

G1 G1

G1

Sub-G1 S

25mgmL

times105

times105times10

5

times105

times105

times105

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

(a)














4752 plusmn 059




4827 plusmn 204



327 plusmn 005

4807 plusmn 078

1755 plusmn 075

3111 plusmn 095

252151050URH (mgmL)

Sub-G1 ()G1 ()S ()G2M ()

(b)




50values at





50







105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI10

3

104

105

106

PI

105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

0

5

10

15

20

Apop

totic

cells

()

(b)



(+) (+) (+)

(+) (+)


V1-L V1-R V1-L V1-R

500 500 235 765 289 711

319 681 200 800

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000C

ount

0

500

1000

Cou

nt

1mgmL

15mgmL 2mgmL

0mgmL 05mgmL

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity10

310

510

710

1

ROS intensity

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowast

05 10 15 2000URH (mgmL)

80

100

120

140

160

180

Relat

ive R

OS

(+) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt



938 62 944 56 900 100

881 119878 122 849 152

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

(a)

lowastlowast


lowastlowast

05 10 15 20 2500URH (mgmL)

80

100

120

140

160

180

200

220

240

260

280

Relat

ive m

itoSO

X (+

) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

80

84

88

92

96

100

MM

P (+

) (

)

(b)


119901





50values of fish



5 Conclusion



Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










2(3) was



Ca9-22CAL 27

lowastlowast

lowast lowastlowast




lowastlowast

lowastlowast

lowastlowast

025 050 075 100 150 200 250000URH (mgmL)

0

20

40

60

80

100

120

ATP

cont

ent (

)


119901



3 Results








25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL








4 Discussion



Sub-G1G2M

S

G1

Sub-G1 S Sub-G1G2M

S

G1

Sub-G1 S Sub-G1G2M

G2M

G2M G2M

S

G1

G1 G1

G1

Sub-G1 S

25mgmL

times105

times105times10

5

times105

times105

times105

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

(a)














4752 plusmn 059




4827 plusmn 204



327 plusmn 005

4807 plusmn 078

1755 plusmn 075

3111 plusmn 095

252151050URH (mgmL)

Sub-G1 ()G1 ()S ()G2M ()

(b)




50values at





50







105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI10

3

104

105

106

PI

105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

0

5

10

15

20

Apop

totic

cells

()

(b)



(+) (+) (+)

(+) (+)


V1-L V1-R V1-L V1-R

500 500 235 765 289 711

319 681 200 800

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000C

ount

0

500

1000

Cou

nt

1mgmL

15mgmL 2mgmL

0mgmL 05mgmL

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity10

310

510

710

1

ROS intensity

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowast

05 10 15 2000URH (mgmL)

80

100

120

140

160

180

Relat

ive R

OS

(+) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt



938 62 944 56 900 100

881 119878 122 849 152

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

(a)

lowastlowast


lowastlowast

05 10 15 20 2500URH (mgmL)

80

100

120

140

160

180

200

220

240

260

280

Relat

ive m

itoSO

X (+

) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

80

84

88

92

96

100

MM

P (+

) (

)

(b)


119901





50values of fish



5 Conclusion



Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL








4 Discussion



Sub-G1G2M

S

G1

Sub-G1 S Sub-G1G2M

S

G1

Sub-G1 S Sub-G1G2M

G2M

G2M G2M

S

G1

G1 G1

G1

Sub-G1 S

25mgmL

times105

times105times10

5

times105

times105

times105

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

(a)














4752 plusmn 059




4827 plusmn 204



327 plusmn 005

4807 plusmn 078

1755 plusmn 075

3111 plusmn 095

252151050URH (mgmL)

Sub-G1 ()G1 ()S ()G2M ()

(b)




50values at





50







105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI10

3

104

105

106

PI

105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

0

5

10

15

20

Apop

totic

cells

()

(b)



(+) (+) (+)

(+) (+)


V1-L V1-R V1-L V1-R

500 500 235 765 289 711

319 681 200 800

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000C

ount

0

500

1000

Cou

nt

1mgmL

15mgmL 2mgmL

0mgmL 05mgmL

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity10

310

510

710

1

ROS intensity

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowast

05 10 15 2000URH (mgmL)

80

100

120

140

160

180

Relat

ive R

OS

(+) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt



938 62 944 56 900 100

881 119878 122 849 152

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

(a)

lowastlowast


lowastlowast

05 10 15 20 2500URH (mgmL)

80

100

120

140

160

180

200

220

240

260

280

Relat

ive m

itoSO

X (+

) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

80

84

88

92

96

100

MM

P (+

) (

)

(b)


119901





50values of fish



5 Conclusion



Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Sub-G1G2M

S

G1

Sub-G1 S Sub-G1G2M

S

G1

Sub-G1 S Sub-G1G2M

G2M

G2M G2M

S

G1

G1 G1

G1

Sub-G1 S

25mgmL

times105

times105times10

5

times105

times105

times105

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

5 10 150DNA content

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

0

100

200

300

400

Cou

nt

(a)














4752 plusmn 059




4827 plusmn 204



327 plusmn 005

4807 plusmn 078

1755 plusmn 075

3111 plusmn 095

252151050URH (mgmL)

Sub-G1 ()G1 ()S ()G2M ()

(b)




50values at





50







105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI10

3

104

105

106

PI

105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

0

5

10

15

20

Apop

totic

cells

()

(b)



(+) (+) (+)

(+) (+)


V1-L V1-R V1-L V1-R

500 500 235 765 289 711

319 681 200 800

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000C

ount

0

500

1000

Cou

nt

1mgmL

15mgmL 2mgmL

0mgmL 05mgmL

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity10

310

510

710

1

ROS intensity

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowast

05 10 15 2000URH (mgmL)

80

100

120

140

160

180

Relat

ive R

OS

(+) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt



938 62 944 56 900 100

881 119878 122 849 152

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

(a)

lowastlowast


lowastlowast

05 10 15 20 2500URH (mgmL)

80

100

120

140

160

180

200

220

240

260

280

Relat

ive m

itoSO

X (+

) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

80

84

88

92

96

100

MM

P (+

) (

)

(b)


119901





50values of fish



5 Conclusion



Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI

103

104

105

106

PI10

3

104

105

106

PI

105

106

104

Annexin V10

510

610

4

Annexin V10

510

610

4

Annexin V

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

0

5

10

15

20

Apop

totic

cells

()

(b)



(+) (+) (+)

(+) (+)


V1-L V1-R V1-L V1-R

500 500 235 765 289 711

319 681 200 800

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000C

ount

0

500

1000

Cou

nt

1mgmL

15mgmL 2mgmL

0mgmL 05mgmL

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity10

310

510

710

1

ROS intensity

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowast

05 10 15 2000URH (mgmL)

80

100

120

140

160

180

Relat

ive R

OS

(+) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt



938 62 944 56 900 100

881 119878 122 849 152

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

(a)

lowastlowast


lowastlowast

05 10 15 20 2500URH (mgmL)

80

100

120

140

160

180

200

220

240

260

280

Relat

ive m

itoSO

X (+

) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

80

84

88

92

96

100

MM

P (+

) (

)

(b)


119901





50values of fish



5 Conclusion



Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(+) (+) (+)

(+) (+)


V1-L V1-R V1-L V1-R

500 500 235 765 289 711

319 681 200 800

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000

Cou

nt

0

500

1000C

ount

0

500

1000

Cou

nt

1mgmL

15mgmL 2mgmL

0mgmL 05mgmL

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity10

310

510

710

1

ROS intensity

103

105

107

101

ROS intensity10

310

510

710

1

ROS intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowast

05 10 15 2000URH (mgmL)

80

100

120

140

160

180

Relat

ive R

OS

(+) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt



938 62 944 56 900 100

881 119878 122 849 152

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

(a)

lowastlowast


lowastlowast

05 10 15 20 2500URH (mgmL)

80

100

120

140

160

180

200

220

240

260

280

Relat

ive m

itoSO

X (+

) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

80

84

88

92

96

100

MM

P (+

) (

)

(b)


119901





50values of fish



5 Conclusion



Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(+) (+) (+)

(+) (+) (+)

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt

0

400

800

Cou

nt



938 62 944 56 900 100

881 119878 122 849 152

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

103

105

107

101

MitoSOX intensity10

310

510

710

1

MitoSOX intensity10

310

510

710

1

MitoSOX intensity

(a)

lowastlowast


lowastlowast

05 10 15 20 2500URH (mgmL)

80

100

120

140

160

180

200

220

240

260

280

Relat

ive m

itoSO

X (+

) (

)

(b)



(+) (+) (+)

(+) (+) (+)

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

80

84

88

92

96

100

MM

P (+

) (

)

(b)


119901





50values of fish



5 Conclusion



Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(+) (+) (+)

(+) (+) (+)

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

0

500

1000

1500

Cou

nt

25mgmL

05mgmL 1mgmL

15mgmL 2mgmL

0mgmL

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

103

105

107

101

MMP intensity10

310

510

710

1

MMP intensity10

310

510

710

1

MMP intensity

(a)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

05 10 15 20 2500URH (mgmL)

80

84

88

92

96

100

MM

P (+

) (

)

(b)


119901





50values of fish



5 Conclusion



Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




50values of fish



5 Conclusion



Competing Interests


Acknowledgments


References






















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article roe protein hydrolysates of giant grouper...

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