Asian Pacific Journal of Cancer Prevention, Vol 13, 2012
1349
DOI:http://dx.doi.org/10.7314/APJCP.2012.13.4.1349 Up-regulation
of Thy-1 Promotes Invasion and Metastasis of Hepatocarcinomas
Asian Pacific J Cancer Prev, 13, 1349-1353
Introduction
Hepaticarcinoma is the fifthmost common
cancerandoneoftheleadingcausesofcancerdeathworldwide.EspeciallywithquickdevelopmentofliverdiseaseduetohepatitisBvirusinfection,incidenceofhepaticarcinomahasbeenontheriseandearlierrecurrenceafteroperationorlivertransplantiscommon.Theunderlyingrootcauseistheexistenceofcancerstemcells(CSCs)(Shupeetal.,2005;Selletal.,2008;Alisonetal.,2009;Chibaetal.,2009;Leeetal.,2009),which,despitetheirsmallnumber,play
amajor role in tumor origination,
development,invasionandearliermetastasis(Reyaetal.,2001;Couzinetal.,2003).Ovalcellsarethestemcellsofliver(Birdetal.,2008;Strick-Marchandetal.,2008;Okabeetal.,2009;Shupeetal.,2009).Recentlyagreatdealofresearchsuggestedapositiverelationshipbetweenitsproliferationand
the incidence of hepaticarcinoma (Alison et
al.,2005;Alisonetal.,2006;Wuetal.,2006).Thy-1,asamarkerproteinofovalcells,hasrecentlybeensuggestedtoplayaroleinpotentiallivercancerstemcells(Dahlkeetal.,2003;Ceafalanetal.,2005;Massonetal.,2006;Yang
et al., 2008;Yang et al., 2008;Xu et al., 2009).As a gene expressed
during embronic period or
underpathologicstate,AFP(alphafetoprotein)participatesinDepartment
of Hepatology Center, Fuzhou General Hospital, Nanjing Military
Area Command, Fuzhou, China *For correspondence:
[email protected]
Abstract
Increasing evidence has revealed that thy-1 was a potential stem
cell marker of liver cancer, but no data have been shown on how
thy-1 regulates the pathophysiology of liver cancer, such as
proliferation, apoptosis, invasion and migration. We previously
demonstrated that thy-1 was expressed in about 1% of hepg2 cells,
thy-1+ hepg2 cells, but not thy-1-, demonstrating high
tumorigenesis on inoculation 0.5x105 cells per BACA/LA mouse after
2 months. In the present study, our results showed that higher
expression of thy-1 occurs in 72% (36/50 cases) of neoplastic
hepatic tissues as compared to 40% (20/50 cases) of control
tissues, and the expression of thy-1 is higher in poorly
differentiated liver tumors than in the well-differentiated ones.
In addition, thy-1 expression was detected in 85% of blood samples
from liver cancer patients, but none in normal subjects or patients
with cirrhosis or hepatitis. There was a significant negative
correlation between thy-1expression and E-cadherin expression (a
marker of invasion and migraton), but not between thy-1 expression
and AFP expression in all the liver cancer and blood samples. We
further investigated the relationship between thy-1 and E- cadherin
in liver cancer hepg2 cell line which was transfected with
pReceiver-M29/thy-1 eukaryotic expression vector followed by
aspirin treatment. Lower expression of E- cadherin but higher
expressions of thy-1 were detected in hepg2 cells transfected with
pReceiver-M29/thy-1. Taken together, our study suggested that thy-1
probably regulates liver cancer invasion and migration. Keywords:
Invasion-migration-thy-1-hepatocarcinomas
RESEARCHCOMMUNICATION
Up-regulation of Thy-1 Promotes Invasion and Metastasis of
HepatocarcinomasBian-Qiao Cheng, Yi Jiang*, Dong-Liang Li,
Jing-Jing Fan, Ming Ma
embryonicdevelopmentandcelldivisionprocess(Kanget al.,
2006).Recent research suggested (Kuhlmann etal., 2006)AFPpossessed
stem cell characteristics
andcouldberegardedasanidealmarkerpredictingearlierliver cancer
recurrence after operation.However, theshortcoming ofAFP loss
expression in some
samplesrequiredamoreaccurateandspecificmarkertopredictlivercanceroccurrenceandrecurrenceindependentoforincombinationwithAFPmarker.E-cadherin,amemberofcadherinfamily,mediatescell-celladhesionsanditslossoffunctioncouldcausebreakdownofcell-cellconjunctions.It
is therefore commonlyused to predict invasion andmetastasis of
variouskindsofmalignant
tumor.Basedonfindingsabove,thisarticlewasaimedtoinvestigatetherelationshipbetweenthy-1,AFPandE-cadherinandtounravelthefunctionofthy-1inhepaticarcinoma.
Materials and Methods
Cell lines
Thehumancelllinehepg2(ATCC,American)wasmaintainedinRPMI1640medium(Hyclone,American)containing10%fetalbovineserum(Hyclone,American)and1%penicillinat37°Cinahumidifiedincubatorwith5%CO2.
Bian-Qiao Cheng et al
Asian Pacific Journal of Cancer Prevention, Vol 13, 20121350
Patients and sample collection Fifty paraffin-embedded
tissueswith liver cancerwho had undergone laparotomy (46/50 had
curativehepaticresectionand4/50hadlivertransplation;18/50well-differentiated
and 32/50 poorly differentiated)were included in the study.
Pathology diagnosiswasmade according to the histology of tumor
specimensand examinations were performed by
experiencedpathologists.Fiftycorrespondingperi-tumoroushealthytissueswere
used as controlswhichwere
confirmedaccordingtopathologicaldiagnosis.ThemajorityofHCC(53cases)andcirrhosisorhepatitispatients(36cases)werepositiveforserumhepatitisBsurfaceantigen.AllthebloodsamplestudieswereapprovedbytheInstitutionalReviewBoardofthefuzhougeneralhospital.
Immunohistochemistry and Immunocytochemistry Immunohistochemical
evaluation on 4mm-thintissue sections from formalin-fixed and
paraffin-embeddedsampleswasperformedforthy-1(sc-53456,dilution1:200;
SantaCruzBiotechnology Inc., SantaCruzCA),AFP(sc-51506,
dilution1:200;
SantaCruzBiotechnologyInc.,SantaCruzCA;),E-cadherin(sc-8426,dilution1:200;
SantaCruzBiotechnology Inc.,
SantaCruzCA).Thestainingreactionswereinterpretedinthepresenceoftissuemastcellsinportalfieldsoftheliverasinternalcontrols,whileAFPandE-cadherinasexternalcontrolsforembryonictissuesandbreastductcarcinomarespectively.
Stainingwasperformedaccordingaspermanufacturer’sinstructions of
vltrasensitiveTMS-P (mouse/rabbit) kit(Maixin, Fujian China).
Briefly, paraffin-embeddedsectionswere deparaffinized and
rehydrated throughgraded alcohols to phosphate buffered saline
(PBS).0.01Mcitratebuffersolution(pH6.0)wasusedinantigenrecovery.Aftersequentialincubationwith3%endogenousperoxidaseblockingsolutionand1%normalnonimmuneserum,
10min for each at room temperature,
primaryantibodieswereincubatedovernightat4°C.Anti-mouseIgGwasusedinsteadofprimaryantibodyasadditionalcontrol.Other
negative controls included theomissionof either primaryor secondary
antibodies.Nonspecificreactionsweredetectedinthenegativecontrols.AccordingtoFromowitzscorestandard,whenthereweredifferentdifferentiationdegreeinthesamesample,predominateareawasselected(Fromowitzetal.,1987)accordingtopositivestainingpercent,scorewasclassifiedasfollows:0:
≤ 25%; 1: 26%~50%; 2: 51%~75%; 3: > 75%. Inaddition, staining
intensitywas rated as 0 (negative),1 (weakly positive), 2
(intermediate) and 3
(stronglypositive).Finally,asummationofthetwoscoresabovewas
furtherclassified
into:0:negative;1~2:“+”;3~4:“++”;5~6:“+++”(Jawharietal.,1997).
Immunocytochemistry HepG2 cellswere plated at 100,000 cells
perwellonenightbeforetheexperiment.BreastcarcinomacelllineDBA-DA231
andun-transfected hepg2 cellswereused as positive and negative
controls respectively.Hepg2cellswere transfectedwithpReceiver-M29/
thy-1expressionvectorandtreatedwithAspirininthetest
group according to themanufacturer’s instructions
ofvltrasensitiveTMS-P(mouse/rabbit)kit(Maixin,FujianChina).Briefly,cellswerefixedin4%paraformaldehydefor10minandpermeabilizedwith0.3%Triton-100for10min.Thefollowingstepswereperformedinthesamewayasimmunohistochemistry.
Immunosorbent assay
Thy-1,AFPandE-cadherinELISA.wereperformedusingELISAkit(R&D)aspermanufacturer’sinstructions.Briefly,50µlstandardor50µlof1:5dilutedsampleswasdispensedtostandardorsamplewells,respectively.Thrityminutesaftertheadditionof50µlenzymelabelreagentat37°C,chromogenicagentAandBwereaddedtoreactfor10minat37°Cwithprotectionfromlight,followingwhichstopbufferwasadded.Thereafter,theabsorbanceat450nmwasmeasuredwithanenzyme-labelinginstrument(ELX-800type).
RT-PCR Experiments were performed using RT-PCR
kit(TakaRa,code:DRRO14A).TotalRNAwasisolatedwithtrizol reagent
(Invitrogen,Cat: 15596-026) accordingto themanufacturer’s
instructionsusing1ml trizol per1*107 cells.TotalRNA
concentrationwas quantifiedwith eppendorf Bio photometer. The
cDNAsweregenerated from 5 µg of totalRNAper sample using20µl
reaction containing 1µl random6 primers,
1µldNTPmixture,0.5µlprimeScripTMRtaseand0.5µlRNaseinhibitor.Gene-specificprimesforthy-1(primerA:5’-AAGGTGACCAGCCTAACGG-3’,primerB:5’-CCCTCGTCCTTGCTAGTGAA-3’),forβ-actin(primerA:
5’-GGAAATCGTGCGTGACATT -3’,
primerB:5’-CGTCATACTCCTGCTTGCTG-3’),andforE-cadherin(primerA:5’-CGCCGACGAGAGCTACACGTTCA-3’,primerB:5’-CCCAGGCGTAGACCAAGAAA-3’)weredesignedusing
the primer 3 software and
synthesized(ShanghaiBiologiccompany,China).
Statistical analysis Descriptive statistics, t-test, χ2 test
andOne-wayANOVAwithLSDmultiplecomparisonwereperformed.Fisherpreciseprobabilisticmethodwasusedtoanalyzeexpressionintensity,Spearmanforcorrelationanalysis.SPSS
software (version 11.5 forWindows; spss
Inc.,Chicago,IL)wasusedforallstatisticalanalyses.Pvaluelessthan0.05wasconsideredsignificant.
Results
Altered expression pattern of thy-1,AFP andE-cadherin in liver
cancers.
Immunohistochemicalstainingshowedreactivitiesofthy-1,AFPandE-cadherinin
40%, 20%, and30%of peritumoral normal
tissues,whereas72%,76%and64%ofneopastic liver
tissuesrespectively.Staining intensities for thy-1 andAFPin normal
peritumoral samples were significantlyweaker than in
tumorsamples,butE-cadherinstainingshowedtheoppositeresults(Figure1).Inaddition,wedetectedasignificantcorrelationbetweenthedegreeofdifferentiationofhepatocarcinomasandtheexpressionof
Asian Pacific Journal of Cancer Prevention, Vol 13, 2012
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DOI:http://dx.doi.org/10.7314/APJCP.2012.13.4.1349 Up-regulation
of Thy-1 Promotes Invasion and Metastasis of Hepatocarcinomas
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Figure 1. Immunohistochemical Staining of Thy-1, AFP and
E-cadherin in Hepatocarcinoma and Peritumoral Normal Liver Samples.
(a) Peritumoral normal tissuesdemonstratingweakAFP reactivity
(magnification 200x).(b)Hepatocarcinoma exhibiting strong
expression ofAFP(magnification200x). (c)Hepatocarcinoma exhibiting
strongexpression of thy-1 (magnification 200x). (d)
Peritumoralnormal liver tissues revealing intermediate thy-1
expression(magnification 100x). (e) Liver cancer cells
demonstratingweaker expressionofE-cadherin (black arrow) compared
tohigherexpressioninnormaltissues(redarrow)(magnification200x).(f)BreastcancerascontrolexhibitingstrongexpressionofE-cadherin(magnification200x)
Figure 2. RT-PCR Analysis of Thy-1 mRNA and E-cadherin mRNA
Expression.
(a)Expressionofthy-1wasincreasedinpReceiver-M29/Thy-1transfectedhepg2cellsbutdramaticallyreducedaftertreatmentwithAspirin.(b)E-cadherinexpressionwaslowestininpReceiver-M29/Thy-1transfectedhepg2cellsbutslightincreasedwithAspirintreatment
Figure 3. Immuocytochemical Staining for E-cadherin in Hepg2
Cells after Differenct Treatments.
(a)E-cadherinstaininginnon-transfectedhepg2cells(magnification200x).(b)ObviouslyweakerstainingforE-cadherininhepg2cells
transfectedwith pReceiver-M29/thy-1 (magnification200x). (c)
Slightlyweaker staining in pReceiver-M29/thy-1hepg2 transfected
cells treatedwithAspirin
(magnification200x).(d)E-cadherinstaininginbreastcancerDAB-DA231cells(magnification200x)
Table 2 Correction Between thy-1 and AFP, E-cadherin by
Immunohistochemistry Proteinofinterest Thy-1 rs p + _
AFP + 27 7 0.116 0.421 _ 11 5 E-cadherin + 18 16 -0.336 0.117 _
14 2
Table 1 Immunohistochemical Reactivity in Liver Cancers and
Peritumoral Nomal Tissues proteinofinterest
tumorperitumousWellPoorly -differentiateddifferentiated
(n=50)(n=50)(n=18)(n=32)
Thy-1 36 20 7 26AFP 38 10 7 22E-cadherin 32 15 15 17
Table 3 Correction Between thy-1 and AFP, E-cadherin by ELISA
groups Serum(U/ml)rsp
Thy-1 3.694 2.961 E-cadherin 1.591 -0.334 0.027 0.696 AFP 3.121
0.046 0.746 1.965
thy-1,AFPandE-cadherin,wherebypoorlydifferentiatedhepatocarcinomasweremorelikelytoexpressthy-1andAFP
but less likely to expressE-cadherin thanwell-differentiated ones
(poorly differentiated: thy-1,AFPandE-cadherinexpressed
in81.2%,68.8%and53.1%ofsamplesrespectively;well-differentiated:thy-1,AFPandE-cadherinexpressedin38.8%,38%and83.3%ofsamplesrespectively)(Table1).
Lack of correlation between thy-1 andAFP, butnegative correlation
between thy-1 and E-cadherin.
Bothpositiveexpressionsamplesornegativefor thy-1andAFPwas 27or
5, Positive expression samples
forthy-1of7casesbutnegative1forAFPandnegative11forthy-1butpositiveforAFPwasalsoobserved,therewas
no significant difference between them (rs=0.116,p=0.421).Both
positive expression samples for
thy-1andE-cadherinwas18,bothnegative for themwas2,positive for
thy-1was 16 but negative
forE-cadherinwas14,negativecorrelationwasdetectedbetweenthem(rs=-0.336,p=0.017)(Table2).Inaddition,from53livercancerserumsamples,levelsofthy-1,AFPandE-cadherindetectedwere3.6942.961,3.1211.965,and1.5910.696respectively.No
correlationwas found
betweenAFPandthy-1(rs=0.046,p=0.746),buttherewasanegativecorrelation
between thy-1 andE-cadherin (rs=-0.334,p=0.027)(Table3).
EnhancedThy-1 expression reduces E-cadherinlevel. In order to
further understand the relationship
Asian Pacific Journal of Cancer Prevention, Vol 13, 2012
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DOI:http://dx.doi.org/10.7314/APJCP.2012.13.4.1349 Up-regulation
of Thy-1 Promotes Invasion and Metastasis of Hepatocarcinomas
early invasion/metastasis of liver cancer are
complexprocesses.Therefore, understanding detailed actionsof thy-1
inhepatocarcinomaanddesigning therapeuticstrategies such asRNAi or
gene knock-out
directedtowardsthy-1wouldholdpromiseforfuturelivercancertherapy.
Acknowledgements
ThebreastcarcinomaDAB-DA231cellswerekindlyprovidedbyQiao-JiaHuangofDepartmentofLaboratoryInstitute,
FuzhouGeneralHospital,NanjingMilitaryDistrict,Fuzhou,China.
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