Research Article Comparative Proteomic Analysis of Human...

Research ArticleComparative Proteomic Analysis of HumanCholangiocarcinoma Cell Lines S100A2 as a PotentialCandidate Protein Inducer of Invasion

Kasima Wasuworawong1 Sittiruk Roytrakul2 Atchara Paemanee2

Kattaleeya Jindapornprasert1 and Waraporn Komyod1

1Department of Biochemistry Faculty of Science Mahidol University 272 Rama VI Road Bangkok 10400 Thailand2Proteomics Research Laboratory Genome Institute National Science and Technology Development Agency111 Thailand Science Park Phahonyothin Road PathumThani 12120 Thailand

Correspondence should be addressed to Waraporn Komyod warapornkommahidolacth

Received 7 November 2014 Accepted 9 March 2015

Academic Editor Joy Scaria

Copyright copy 2015 Kasima Wasuworawong et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Cholangiocarcinoma (CCA) is a bile duct cancer commonly found in Asia including Thailand and especially in the northeasternregion of Thailand To identify the proteins involved in carcinogenesis and metastasis of CCA protein expression profiles ofhigh-invasive KKU-M213 and low-invasive KKU-100 cell lines were compared using a comparative GeLC-MSMS proteomicsanalysis A total of 651 differentially expressed proteins were detected of which 27 protein candidates were identified as havingfunctions involved in cell motility A total of 22 proteins were significantly upregulated in KKU-M213 whereas 5 proteins weredownregulated in KKU-M213 S100A2 a calcium-binding protein in S100 protein family is upregulated in KKU-M213 S100A2is implicated in metastasis development in several cancers The protein expression level of S100A2 was verified by Western blotanalysis Intriguingly high-invasive KKU-M213 cells showed higher expression of S100A2 than KKU-100 cells consistent withproteomic data suggesting that S100A2 may be a key protein involved in the progression of CCA However the biological functionof S100A2 in cholangiocarcinoma remains to be elucidated S100A2 might be a potential biomarker as well as a novel therapeutictarget in CCA metastasis

1 Introduction

Cholangiocarcinoma (CCA) is a malignant tumor thatoriginates from epithelial cells of the bile duct CCA isdifficult to diagnose and the curative treatment remainschallenging Due to its late clinical manifestation morbidityand mortality rates of CCA are high and its incidence hasbeen increasing over the past three decades especially innortheastern Thailand [1] CCA is often associated withmetastasis which is a highly complicated process that involvescell motility invasion angiogenesis intravasation of tumorcells into the blood stream and finally extravasation and col-onization of tumor cells at secondary sites [2] The migrationand invasion properties have been a hallmark of cancer[3] including CCA in incrimination of disease severity Inparticular metastasis is one of the major hindrances to

the treatment of CCA and many cancer types that causemore than 90 of cancer-associated mortality [4] MoreoverCCA is resistant to radio- and chemotherapy and surgicalresection is the only effective therapy against this typeof cancer [5ndash7] Hence understanding the mechanism ofinvasion and metastasis will be important in identifying keyplayers involved which may lead to development of effectivetargeted therapy against this deadly disease

Here we compared the protein profiles of two humanCCA cell lines with different metastatic abilities KKU-M213and KKU-100 KKU-M213 a high-invasive cell line origi-nated from adenosquamous CCA with well differentiationand KKU-100 a low-invasive cell line was isolated fromadenocarcinomaCCAwith poor differentiation [8] Studyingthe differential protein patterns of these cell lines allowedus to identify several proteins which might be the key

Hindawi Publishing CorporationDisease MarkersVolume 2015 Article ID 629367 6 pageshttpdxdoiorg1011552015629367

2 Disease Markers

determinant of themetastatic properties of the CCA cells andmight be beneficial as a future drug target

Proteomics analysis is currently considered to be apowerful tool for global evaluation of protein expressionand proteomics has been widely applied in analysis ofdiseases especially in fields of cancer research In this studywe employed a comparative SDS-PAGE coupled with LC-MSMS (GeLC-MSMS) based proteomics approach [9] tocompare the protein expression profile of the high-invasiveKKU-M213 cell line with low-invasive KKU-100 cell line tobetter understand the development and metastasis of CCAMSMS spectra of obtained proteins were identified based onNCBI human database This technique can identify potentialcandidate proteins that might be involved in the differentdegrees of invasiveness displayed by the two CCA cell linesDifferential expression at transcription and protein expres-sion levels of a candidate protein was further confirmed byquantitative real-time PCR and Western blot analysis

2 Materials and Methods

21 Cell Culture Human cholangiocarcinoma cell linesKKU-M213 and KKU-100 were kindly provided by ProfessorBanchob Sripa (Khon Kaen University Khon Kaen Thai-land) Cells were cultured in Hamrsquos F-12 nutrient mixturemedium (Invitrogen Corp Auckland NZ) supplementedwith 10 fetal bovine serum (FBS) 100UmL penicillin100 120583gmL streptomycin sulfate (Invitrogen Corp AucklandNZ) and 15mM HEPES (USB Corp OH USA) Cells wereincubated at 37∘C in a humidified atmosphere with 5 CO

2

22 Invasion Assay Invasion assay was determined byMatrigel transwell in vitro invasion assay as previouslydescribed [10] with some modification In brief the upperchamber of a transwell unit (65 mm diameter polycarbonatemembrane with 8 120583m pore size) (Corning Incorporated LifeScience Corning NY) was coated with 30120583g of Matrigel(BD Biosciences Bedford MA) Cells (80 confluent) wereharvested using 025 Trypsin-EDTA (Invitrogen CorpAuckland NZ) and resuspended in serum-free media A200120583L aliquot of cell suspension (105 cells) was added to theupper chamber The lower chamber was filled with 600 120583Lof media containing 1 FBS After 12 hours of incubationat 37∘C under CO

2atmosphere noninvading cells in the

upper chamber were removed and cells that invaded theMatrigel andhad attached to the lower surface of the transwellmembrane were fixed with 25 methanol for 15min andstainedwith 05 crystal violet Invaded cells were counted in5 random fields under light microscope at 10x magnificationThe reported values represent mean plusmn SE of the resultsobtained from three independent experiments

23 Preparation of Cell Lysates Cells were washed twicewith cold PBS containing 100 120583M Na

3VO4 trypsinized and

collected by centrifugation Cell pellets were kept in minus80∘Cprior to use The pellets were lysed in Tris-lysis buffer(20mMTris-HCl pH 75 150mMNaCl 10mMNaF 1 (vv)Triton-X) supplemented with 1mM Na

3VO4 1 mM PMSF

and protease inhibitors (Sigma UK) and chilled on ice for30min Lysates were centrifuged at 13000 rpm for 10min at4∘C to discard cell debris and protein concentration in thesupernatant was determined by Lowry assay [11] The celllysates were stored at minus20∘C

24 One-Dimension Gel Electrophoresis and Tryptic In-GelDigestion Cell lysates (30 120583g) were mixed in loading buffer(3125mMTris-Cl pH 68 50 glycerol 10 SDS 005 bro-mophenol blue 125 2-B-mercaptoethanol) and boiled for5min before being applied on a 125 SDS-polyacrylamidegel (BioRad Hercules CA) using Hoefer apparatus AfterCoomassie blue staining gel slices were excised cut into1mm3 cubes and subjected to in-gel tryptic digestion Theexcised gel slices were reduced with 10mM DTT10mMNH4HCO3 alkylated with 100mM IAA10mM NH

4HCO3

and digested with 1 ng protein per 20 ng sequencing gradetrypsin (Promega Germany) at 37∘C overnight

25 Protein Identification Using LC-MSMS Tryptic peptideswere protonated with 01 formic acid before injection intoNanoAcquity system (Waters Corp Milford MA) equippedwith a Symmetry C

185 120583m 180-120583m times 20-mm Trap column

and a BEH130 C18

17 120583m 100-120583m times 100-mm analyticalreversed phase column (Waters Corp Milford MA) [Glu1]fibrinopeptide B was used as the reference sprayer of theNanoLockSpray source of the mass spectrometer Analysisof tryptic peptides was performed using a SYNAPT HDMSmass spectrometer (Waters Corp Manchester UK) Thetime-of-flight analyzer of the mass spectrometer was exter-nally calibrated with [Glu1] fibrinopeptide BThe quadrupolemass analyzer was adjusted such that ions from 119898119911 300 to1800 were efficiently transmitted BSA used for normaliza-tion was performed along with the samples

MS intensities of individual LC-MS analysis were dif-ferentially quantified by using DeCyder MS DifferentialAnalysis Software (GE Healthcare USA) PepMatch modulewas used for evaluating the average abundance ratio of eachsample peptide allowing for automated detection of peptidesand assignment of charge states The MSMS data wassearched against the NCBInr database and identified by usingMascot software (Matrix Science London UK) Database in-terrogation was implemented as follows taxonomymdashhomosapiens databasemdashNCBInr enzymemdashtrypsin fixed modifi-cationmdashcarbamidomethyl variable modificationmdashoxidationof methionine residues mass valuesmdashmonoisotopic peptidemass tolerancemdash2Da peptide charge statemdash1+ 2+ and3+ Protein accession numbers were classified according totheir biological function by PANTHER Classification systemversion 81 (httpwwwpantherdborggeneListAnalysisdo)

26 Western Blot Analysis A total of 30 120583g of protein lysateswas separated by 12 SDS-PAGE and then transferred toPVDFmembrane (Pall Germany) by semidry electroblottingat constant voltage (25V) for 60min The membranes wereblocked with 5 BSA in 1x TBS-N for 1 hr and then incubatedwith anti-S100A2 primary antibody (Abcam UK) at 4∘Covernight The blots were washed three times for 5min

Disease Markers 3

with TBS-N buffer and incubated with anti-rabbit HRP-conjugated secondary antibody (Santa Cruz BiotechnologyInc Santa Cruz CA) at room temperature for 30min Signalswere detected using Amersham ECL PrimeWestern BlottingDetection Reagent (GE Healthcare USA)

27 Quantitative Real-Time PCR Total RNA was extractedusing illustra RNAspin Mini kit (GE Healthcare USA)as described by the manufacturer 1 120583g of total RNA wasconverted to cDNA using ImProm-II Reverse TranscriptionSystem kit (Promega Germany) using random hexamerprimers according to the manufacturerrsquos descriptionThe PCR reaction was performed in a final volume of 20120583Lcontaining 100 ng of cDNA 300 nMof each primer and 10 120583LFastStart Universal SYBRMaster (Roche Germany) Specificprimers were as follows S100A2 forward 51015840-CTGGGTCTG-TCTCTGCCACC-31015840 S100A2 reverse 51015840-GCAGGAGTA-CTTGTGGAAGGTAGTG-31015840 and 120573-actin forward 51015840-CTC-TTCCAGCCTTCCTTCCT-31015840 120573-actin reverse 51015840-AGCACT-GTGTTGGCGTACAG-31015840 Thermal cycling conditionswere as follows denaturing at 95∘C for 10min followedby 40 cycles of 95∘C for 15 s and 60∘C for 30 s Real-timePCR was performed on Mx3000P QPCR System (AgilentTechnologies USA) All PCR amplifications were conductedin triplicate The 2minusΔΔCT method [12] was used to calculatethe relative gene expression level

28 Statistical Analysis Statistical analysis was performedusing Studentrsquos 119905-test with 119875 lt 005 considered to besignificant

3 Results and Discussion

Cell invasion using the Boyden-transwell migration assayrevealed that the KKU-M213 cells displayed approximately8-fold higher level of invasiveness than KKU-100 cells(Figure 1) The results were consistent with previous report[13] that KKU-M213 is a high-invasive cell line while KKU-100 is a low-invasive cell line A comparative SDS-PAGE ofprotein lysates of both cell types initially indicated differencesin intensity of protein bands as shown in Figure 2 Uponin-gel tryptic digestions coupled with LC-MSMS (GeLC-MSMS) analysis of the protein lysates of high-invasive KKU-M213 cells and low-invasive KKU-100 cells six hundredand fifty-one differentially expressed proteins were identifiedThese proteins were classified into 8 groups of functionalproteins according to their biological processes synergizedby UniProtKB using PANTHER classification system Theseproteins were categorized as cellular component (32)transcription and translation process (15) metabolic pro-cess (12) signal transduction (11) immune response(10) cell motility (4) unknown (13) and others (3)(Figure 3) Among these 27 proteins were identified belong-ing to the cell motility groupThe relative expression of theseproteins was determined by reciprocal common fractionbetween peptide intensities of the two cell lines With thisapproach a total of 22 proteins were found to be significantly

0

2000

4000

6000

8000

10000

KKU-M213 KKU-100

Num

ber o

f inv

adin

g ce

lls

Cell lines

Figure 1 In vitro invasion assays of KKU-M213 and KKU-100cells were conducted in a transwell unit coated with Matrigel Cellsin serum-free medium were plated in the upper chamber of atranswell unit After 12 hours of incubation cells invading to thelower compartment of the transwell unit were stained and countedThe numbers of invading cells are presented as mean plusmn SE of resultsobtained from three independent experiments 119875 lt 001 comparedwith KKU-100 cell line

KKU

-M21

3

KKU

-100

97kDa

66kDa

45kDa

30kDa

20kDa

144kDa

Figure 2 Differential expression of proteins comparing betweenKKU-M213 and KKU-100 cell lines 30120583g of protein lysates wasseparated in 125 SDS-PAGEwith constant amplitude (20mAgel)The gel was stained with Coomassie blue R-250 to visualize proteinbands

upregulated in KKU-M213 (Table 1) whereas 5 proteins weredownregulated in KKU-M213 (Table 2)

As shown in Table 1 the expression of S100A2 is notablyhigher in KKU-M213 than in KKU-100 The S100A2 proteinis a calcium-binding protein in S100 protein family and hasbeen implicated in the initiation and progression of humancancers such as epithelial ovarian cancer pancreatic cancerand gastric cancer [14 15] and is associated with cancermetastasis process [15ndash17] Further verification of the proteinexpression level of S100A2 by Western blot analysis using anantibody specific to S100A2 confirmed that the expression ofS100A2 inKKU-M213 is obviously higher thanKKU-100 cells

4 Disease Markers

Table 1 Overexpression of proteins in KKU-M213 compared to KKU-100

Protein ID details Sequence Score Folda

gi|5174661 Protein S100A2 ELPSFVGEK 2251 180gi|18088719 Tubulin beta ISVYYNEATGGK 6059 168gi|12667788 Myosin-9 IAQLEEQLDNETK 3310 158gi|4885583 Rho-associated protein kinase 1 SVAMCEMEK 296 147gi|66346662 Rho GTPase-activating protein 8 isoform 1 KDGDLTMWPR 2028 142gi|46249758 Ezrin IALLEEAR 4955 133gi|5174735 Tubulin beta-4B chain INVYYNEATGGK 6496 129gi|116063573 Filamin-A isoform 1 SPFEVYVDK 3632 127gi|4502101 Annexin A1 TPAQFDADELR 9372 124gi|33469929 Pikachurin isoform 1 precursor QKIVEGMAEGGFTQIK 399 123gi|47059046 Protocadherin-23 isoform 1 AVPPRMPAVNLGQVPPK 920 123gi|4501891 Alpha-actinin-1 isoform b AGTQIENIEEDFRDGLK 2013 121gi|336020355 Mitogen-activated protein kinase 4 isoform 2 LTANETQSASSTLQK 1091 121gi|50845388 Annexin A2 isoform 1 GVDEVTIVNILTNR 7932 120gi|28372535 Tctex1 domain-containing protein 3 VQQILTESLK 3094 120gi|40788018 Rho GTPase-activating protein 11A isoform 2 MSSNTEKK 847 118gi|105990514 Filamin-B isoform 2 VLFASQEIPASPFR 4073 115gi|224451128 Protein eyes shut homolog isoform 1 ISDISFHYEFHLK 1375 115gi|122937398 Cytoplasmic dynein 2 heavy chain 1 isoform 2 AADLKDLNSR 1559 113gi|4503355 Dedicator of cytokinesis protein 1 KVTAKIDYGNR 312 110gi|7662284 Protein-methionine sulfoxide oxidase MICAL2 AAHLASMFGHGDFPQNK 1155 108gi|7657532 Protein S100A6 LMEDLDR 3648 108aStatistical significance was determined by Studentrsquos 119905-test (119875 lt 005)

Table 2 Overexpression of proteins in KKU-100 compared to KKU-M213


gi|4504981 Galectin-1 SFVLNLGK 3026 233gi|4506091 Mitogen-activated protein kinase 6 RLDHDNIVK 903 139gi|62548860 Matrilin-2 isoform a precursor NFNSAKDMK 1244 121gi|53828924 Neuropeptides BW receptor type 1 TYSAAR 992 122gi|6005810 Mitogen-activated protein kinase 1 isoform 2 LGGGTYGEVFK 848 112aStatistical significance was determined by Studentrsquos 119905-test (119875 lt 005)

Other3

Cell motility4

Immune response10

Signal transduction11

Metabolic process12Unknown

13

Transcription andtranslation process

15

Cellular component32

Figure 3 Gene ontology pie-charts showed categorization of 651identified proteins fromMSMS spectra according to their biologicalprocesses using the PANTHER classification system

(Figure 4(a)) in compliance with the expression profile ofproteomic analysis Moreover S100A2 had higher expressionin KKU-M213 than MMNK-1 a normal cholangiocyte cellline since S100A2 protein expression was not detected inMMNK-1 cells (data not shown) The expression of S1000A2at transcription level as determined by quantitative real-time PCR also elucidated about 850-fold higher expressionin KKU-M213 than KKU-100 (Figure 4(b)) Altogether theexpression levels of S100A2 in correlation with the invasive-ness of KKU-M213 cells implied that S100A2 might be akey protein involved in the progression of CCA S100A2 hasbeen reported to be a potential biomarker for diagnosis andprognosis in many types of cancer [14] with overexpressionand downregulation in various types of cancer [15 18ndash22]The role of S100A2 in promoting NSCLC metastasis [18] andmigrationinvasion in hepatocellular carcinoma [23] has alsobeen documented The S100A2 expression has been shownto be necessary for TGF-120573-mediated migrationinvasion [23]

Disease Markers 5

Actin

S100A2

KKU

-M21

3

KKU

-100

(42kDa)

(11kDa)

(a)

1200

1000

800

0

KKU-M213 KKU-100Cell lines

Rela

tive S

100

A2

expr

essio

n(n

orm

aliz

ed b

y ch

olan

gioc

yte)

(b)

Figure 4 Validation of S100A2 in cholangiocarcinoma KKU-M213 and KKU-100 cell lines (a) Western blot analysis (b) Quantitative real-time PCR Data are presented as mean plusmn SE of S100A2 mRNA level normalized with 120573-actin mRNA obtained from three independentexperiments 119875 = 0003 compared with KKU-100 cell line

and was regulated by TGF-120573-induced MEKERK signalingEctopic expression of S100A2 also elucidated that S100A2regulated PI3KAkt signaling a potent pathway involved inepithelial-mesenchymal transition (EMT) [24] FurthermoreS100A2 can interact with p53 to modulate its transcriptionalactivity in the calcium-dependent manner [25] while Smad3does not need calcium ion to interact with S100A2 [24]Taken together our findings of the S100A2 differential over-expression in KKU-M213 signified its role in invasive abilityin CCA Recently immunohistochemistry of resected CCAtissues illustrated that S100A2 expression level was correlatedwith severity of CCA cancer progression and suggested it as apotential biomarker for the diagnosis of cholangiocarcinomapatients [26] However the biological function of S100A2toward invasiveness and progression of cholangiocarcinomastill needs further investigations Importantly our resultssuggested that S100A2 could be a candidate biological markerand novel target for diagnosis of CCA metastasis

4 Conclusions

Our study aims to compare the protein profiles of the twoCCA cell lines KKU-M213 and KKU-100 with an attemptto identify proteins associated with invasiveness of CCASDS-PAGE coupled with LC-MSMS (GeLC-MSMS) is apotential initial technique to obtain an entire protein expres-sion profile followed by further verification steps With thismethod we showed a profile of proteome alterations inthe two CCA cells with different invasive ability We haveidentified 651 proteins that were found to be differentiallyexpressed between the two cell lines and could be categorizedinto at least 6 functional groups including cellular component(32) transcription and translation process (15) metabolic

process (12) signal transduction (11) immune response(10) and cell motility (4) In cell motility group S100A2a calcium-binding protein which had pronouncedly 18-fold higher expression in high-invasive KKU-M213 cellshas been identified Higher expression of S100A2 was con-firmed at both transcription and protein expression levelsOur results suggested that S100A2 could be a significantcandidatemarker of CCA carcinogenesis and possibly a noveltherapeutic target inCCAmetastasis Further investigation ofthe biological function of S100A2 in CCA could be pursuedby overexpressing of S100A2 in S100A2-depleted cell lineand by an approach using knock-down protein expression inS100A2-expressing cell line Finally our observations by pro-teomic approach provided useful insights for understandingthe mechanism involved in CCA carcinogenesis and couldhave implications in improved CCA diagnosis and prognosiscapability

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This work was supported by Faculty of Science MahidolUniversity the Office of the Higher Education Commissionand Thailand Research Fund (MRG5380073) The authorswish to thank Associate Professor Pornpimol Rongnoparatfor critical reading of the paper and for her helpful com-ments

6 Disease Markers

References

[1] B Sripa S Kaewkes P Sithithaworn et al ldquoLiver fluke inducescholangiocarcinomardquo PLoS medicine vol 4 no 7 article e2012007

[2] A F Chambers A C Groom and I C MacDonald ldquoDissem-ination and growth of cancer cells in metastatic sitesrdquo NatureReviews Cancer vol 2 no 8 pp 563ndash572 2002

[3] D Hanahan and R AWeinberg ldquoHallmarks of cancer the nextgenerationrdquo Cell vol 144 no 5 pp 646ndash674 2011

[4] W G Stetler-Stevenson ldquoMatrix metalloproteinases in angio-genesis a moving target for therapeutic interventionrdquo Journalof Clinical Investigation vol 103 no 9 pp 1237ndash1241 1999

[5] C D Anderson C W Pinson J Berlin and R S CharildquoDiagnosis and treatment of cholangiocarcinomardquo Oncologistvol 9 no 1 pp 43ndash57 2004

[6] G J Gores ldquoCholangiocarcinoma current concepts andinsightsrdquo Hepatology vol 37 no 5 pp 961ndash969 2003

[7] A E Sirica ldquoCholangiocarcinoma molecular targeting strate-gies for chemoprevention and therapyrdquo Hepatology vol 41 no1 pp 5ndash15 2005

[8] B Sripa S Leungwattanawanit T Nitta et al ldquoEstablishmentand characterization of an opisthorchiasis-associated cholan-giocarcinoma cell line (KKU-100)rdquo World Journal of Gastroen-terology vol 11 no 22 pp 3392ndash3397 2005

[9] S R Piersma M O Warmoes M de Wit I de Reus J CKnol and C R Jimenez ldquoWhole gel processing procedure forGeLC-MSMS based proteomicsrdquo Proteome Science vol 11 no1 article 17 2013

[10] A Albini Y Iwamoto and H K Kleinman ldquoA rapid in vitroassay for quantitating the invasive potential of tumor cellsrdquoCancer Research vol 47 no 12 pp 3239ndash3245 1987

[11] O H Lowry N J Rosebrough A L Farr and R J RandallldquoProtein measurement with the Folin phenol reagentrdquo TheJournal of Biological Chemistry vol 193 no 1 pp 265ndash275 1951

[12] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2(-Delta Delta C(T))methodrdquoMethods vol 25 no 4 pp 402ndash4082001

[13] K Ratthaphol K Imtawil S Wongkham and C WongkhamldquoDevelopment of homemade cholangiocarcinoma DNA arrayrdquoSrinagarind Medical Journal pp 203ndash205 2007

[14] I Salama P S Malone F Mihaimeed and J L Jones ldquoA reviewof the S100 proteins in cancerrdquo European Journal of SurgicalOncology vol 34 no 4 pp 357ndash364 2008

[15] S Wolf C Haase-Kohn and J Pietzsch ldquoS100A2 in canceroge-nesis a friend or a foerdquoAmino Acids vol 41 no 4 pp 849ndash8612011

[16] S K Mishra H R Siddique and M Saleem ldquoS100A4 calcium-binding protein is key player in tumor progression and metas-tasis preclinical and clinical evidencerdquo Cancer and MetastasisReviews vol 31 no 1-2 pp 163ndash172 2012

[17] E Lukanidin and J P Sleeman ldquoBuilding the niche the roleof the S100 proteins in metastatic growthrdquo Seminars in CancerBiology vol 22 no 3 pp 216ndash225 2012

[18] E Bulk B Sargin U Krug et al ldquoS100A2 induces metastasis innon-small cell lung cancerrdquoClinical Cancer Research vol 15 no1 pp 22ndash29 2009

[19] R Yao A Lopez-Beltran G T Maclennan R Montironi JN Eble and L Cheng ldquoExpression of S100 protein familymembers in the pathogenesis of bladder tumorsrdquo AnticancerResearch vol 27 no 5 pp 3051ndash3058 2007

[20] H-L Hsieh B W Schafer N Sasaki and C W HeizmannldquoExpression analysis of S100 proteins and RAGE in humantumors using tissue microarraysrdquo Biochemical and BiophysicalResearch Communications vol 307 no 2 pp 375ndash381 2003

[21] C D Hough K R Cho A B Zonderman D R Schwartz andP JMorin ldquoCoordinately up-regulated genes in ovarian cancerrdquoCancer Research vol 61 no 10 pp 3869ndash3876 2001

[22] D B Villaret T Wang D Dillon et al ldquoIdentification ofgenes overexpressed in head and neck squamous cell carcinomausing a combination of complementary DNA subtraction andmicroarray analysisrdquo Laryngoscope vol 110 no 3 part 1 pp374ndash381 2000

[23] S Naz P Ranganathan P Bodapati A H Shastry L NMishra and P Kondaiah ldquoRegulation of S100A2 expressionby TGF-120573-induced MEKERK signalling and its role in cellmigrationinvasionrdquo Biochemical Journal vol 447 no 1 pp 81ndash91 2012

[24] S Naz M Bashir P Ranganathan P Bodapati V Santoshand P Kondaiah ldquoProtumorigenic actions of S100A2 involveregulation of PI3Akt signaling and functional interaction withSmad3rdquo Carcinogenesis vol 35 no 1 pp 14ndash23 2014

[25] A Muelleri B W Schafer S Ferrari et al ldquoThe Calcium-binding protein S100A2 interacts with p53 and modulates itstranscriptional activityrdquoThe Journal of Biological Chemistry vol280 no 32 pp 29186ndash29193 2005

[26] Y Sato K HaradaM Sasaki and Y Nakanuma ldquoClinicopatho-logical significance of S100 protein expression in cholangiocar-cinomardquo Journal of Gastroenterology andHepatology vol 28 no8 pp 1422ndash1429 2013

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Disease Markers

determinant of themetastatic properties of the CCA cells andmight be beneficial as a future drug target

Proteomics analysis is currently considered to be apowerful tool for global evaluation of protein expressionand proteomics has been widely applied in analysis ofdiseases especially in fields of cancer research In this studywe employed a comparative SDS-PAGE coupled with LC-MSMS (GeLC-MSMS) based proteomics approach [9] tocompare the protein expression profile of the high-invasiveKKU-M213 cell line with low-invasive KKU-100 cell line tobetter understand the development and metastasis of CCAMSMS spectra of obtained proteins were identified based onNCBI human database This technique can identify potentialcandidate proteins that might be involved in the differentdegrees of invasiveness displayed by the two CCA cell linesDifferential expression at transcription and protein expres-sion levels of a candidate protein was further confirmed byquantitative real-time PCR and Western blot analysis

2 Materials and Methods

21 Cell Culture Human cholangiocarcinoma cell linesKKU-M213 and KKU-100 were kindly provided by ProfessorBanchob Sripa (Khon Kaen University Khon Kaen Thai-land) Cells were cultured in Hamrsquos F-12 nutrient mixturemedium (Invitrogen Corp Auckland NZ) supplementedwith 10 fetal bovine serum (FBS) 100UmL penicillin100 120583gmL streptomycin sulfate (Invitrogen Corp AucklandNZ) and 15mM HEPES (USB Corp OH USA) Cells wereincubated at 37∘C in a humidified atmosphere with 5 CO

2

22 Invasion Assay Invasion assay was determined byMatrigel transwell in vitro invasion assay as previouslydescribed [10] with some modification In brief the upperchamber of a transwell unit (65 mm diameter polycarbonatemembrane with 8 120583m pore size) (Corning Incorporated LifeScience Corning NY) was coated with 30120583g of Matrigel(BD Biosciences Bedford MA) Cells (80 confluent) wereharvested using 025 Trypsin-EDTA (Invitrogen CorpAuckland NZ) and resuspended in serum-free media A200120583L aliquot of cell suspension (105 cells) was added to theupper chamber The lower chamber was filled with 600 120583Lof media containing 1 FBS After 12 hours of incubationat 37∘C under CO

2atmosphere noninvading cells in the

upper chamber were removed and cells that invaded theMatrigel andhad attached to the lower surface of the transwellmembrane were fixed with 25 methanol for 15min andstainedwith 05 crystal violet Invaded cells were counted in5 random fields under light microscope at 10x magnificationThe reported values represent mean plusmn SE of the resultsobtained from three independent experiments

23 Preparation of Cell Lysates Cells were washed twicewith cold PBS containing 100 120583M Na

3VO4 trypsinized and

collected by centrifugation Cell pellets were kept in minus80∘Cprior to use The pellets were lysed in Tris-lysis buffer(20mMTris-HCl pH 75 150mMNaCl 10mMNaF 1 (vv)Triton-X) supplemented with 1mM Na

3VO4 1 mM PMSF

and protease inhibitors (Sigma UK) and chilled on ice for30min Lysates were centrifuged at 13000 rpm for 10min at4∘C to discard cell debris and protein concentration in thesupernatant was determined by Lowry assay [11] The celllysates were stored at minus20∘C

24 One-Dimension Gel Electrophoresis and Tryptic In-GelDigestion Cell lysates (30 120583g) were mixed in loading buffer(3125mMTris-Cl pH 68 50 glycerol 10 SDS 005 bro-mophenol blue 125 2-B-mercaptoethanol) and boiled for5min before being applied on a 125 SDS-polyacrylamidegel (BioRad Hercules CA) using Hoefer apparatus AfterCoomassie blue staining gel slices were excised cut into1mm3 cubes and subjected to in-gel tryptic digestion Theexcised gel slices were reduced with 10mM DTT10mMNH4HCO3 alkylated with 100mM IAA10mM NH

4HCO3

and digested with 1 ng protein per 20 ng sequencing gradetrypsin (Promega Germany) at 37∘C overnight

25 Protein Identification Using LC-MSMS Tryptic peptideswere protonated with 01 formic acid before injection intoNanoAcquity system (Waters Corp Milford MA) equippedwith a Symmetry C

185 120583m 180-120583m times 20-mm Trap column

and a BEH130 C18

17 120583m 100-120583m times 100-mm analyticalreversed phase column (Waters Corp Milford MA) [Glu1]fibrinopeptide B was used as the reference sprayer of theNanoLockSpray source of the mass spectrometer Analysisof tryptic peptides was performed using a SYNAPT HDMSmass spectrometer (Waters Corp Manchester UK) Thetime-of-flight analyzer of the mass spectrometer was exter-nally calibrated with [Glu1] fibrinopeptide BThe quadrupolemass analyzer was adjusted such that ions from 119898119911 300 to1800 were efficiently transmitted BSA used for normaliza-tion was performed along with the samples

MS intensities of individual LC-MS analysis were dif-ferentially quantified by using DeCyder MS DifferentialAnalysis Software (GE Healthcare USA) PepMatch modulewas used for evaluating the average abundance ratio of eachsample peptide allowing for automated detection of peptidesand assignment of charge states The MSMS data wassearched against the NCBInr database and identified by usingMascot software (Matrix Science London UK) Database in-terrogation was implemented as follows taxonomymdashhomosapiens databasemdashNCBInr enzymemdashtrypsin fixed modifi-cationmdashcarbamidomethyl variable modificationmdashoxidationof methionine residues mass valuesmdashmonoisotopic peptidemass tolerancemdash2Da peptide charge statemdash1+ 2+ and3+ Protein accession numbers were classified according totheir biological function by PANTHER Classification systemversion 81 (httpwwwpantherdborggeneListAnalysisdo)

26 Western Blot Analysis A total of 30 120583g of protein lysateswas separated by 12 SDS-PAGE and then transferred toPVDFmembrane (Pall Germany) by semidry electroblottingat constant voltage (25V) for 60min The membranes wereblocked with 5 BSA in 1x TBS-N for 1 hr and then incubatedwith anti-S100A2 primary antibody (Abcam UK) at 4∘Covernight The blots were washed three times for 5min

Disease Markers 3






0

2000

4000

6000

8000

10000

KKU-M213 KKU-100

Num

ber o

f inv

adin

g ce

lls

Cell lines


KKU

-M21

3

KKU

-100

97kDa

66kDa

45kDa

30kDa

20kDa

144kDa




4 Disease Markers







Other3

Cell motility4

Immune response10



13


15




Disease Markers 5

Actin

S100A2

KKU

-M21

3

KKU

-100

(42kDa)

(11kDa)

(a)

1200

1000

800

0


Rela

tive S

100

A2

expr

essio

n(n

orm

aliz

ed b

y ch

olan

gioc

yte)

(b)



4 Conclusions





Acknowledgments


6 Disease Markers

References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 3






0

2000

4000

6000

8000

10000

KKU-M213 KKU-100

Num

ber o

f inv

adin

g ce

lls

Cell lines


KKU

-M21

3

KKU

-100

97kDa

66kDa

45kDa

30kDa

20kDa

144kDa




4 Disease Markers







Other3

Cell motility4

Immune response10



13


15




Disease Markers 5

Actin

S100A2

KKU

-M21

3

KKU

-100

(42kDa)

(11kDa)

(a)

1200

1000

800

0


Rela

tive S

100

A2

expr

essio

n(n

orm

aliz

ed b

y ch

olan

gioc

yte)

(b)



4 Conclusions





Acknowledgments


6 Disease Markers

References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















4 Disease Markers







Other3

Cell motility4

Immune response10



13


15




Disease Markers 5

Actin

S100A2

KKU

-M21

3

KKU

-100

(42kDa)

(11kDa)

(a)

1200

1000

800

0


Rela

tive S

100

A2

expr

essio

n(n

orm

aliz

ed b

y ch

olan

gioc

yte)

(b)



4 Conclusions





Acknowledgments


6 Disease Markers

References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Disease Markers 5

Actin

S100A2

KKU

-M21

3

KKU

-100

(42kDa)

(11kDa)

(a)

1200

1000

800

0


Rela

tive S

100

A2

expr

essio

n(n

orm

aliz

ed b

y ch

olan

gioc

yte)

(b)



4 Conclusions





Acknowledgments


6 Disease Markers

References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















6 Disease Markers

References
































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article Comparative Proteomic Analysis of Human...

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