The effect of Helicobacter pylori infection on sonic hedgehog signal pathways in gastric epithelial cells

(1)

Theeffectof

Helicobacterpylori

i

nfecti

ononsoni

chedgehogsi

gnal

pathwaysi

ngastri

cepi

thel

i

alcel

l

s

Yeun Jung Choi

Department of Medical Science

(2)

Theeffectof

Helicobacterpylori

i

nfecti

ononsoni

chedgehogsi

gnal

pathwaysi

ngastri

cepi

thel

i

alcel

l

s

Directed by Professor

YongChanLee

The Master's Thesis

submitted to the Department of Medical Science

the Graduate School of Yonsei University

in partial fulfillment of the requirements for the

degree of Master of Medical Science

Yeun Jung Choi

(3)

This certifies that the Master´s Thesis of

Yeun Jung Choi

_{is approved.}

__________________________________

Thesis Supervisor :

__________________________________

Thesis Committee Member #1

__________________________________

Thesis Committee Member #2

Department of Medical Science

The Graduate School, Yonsei University

(4)

ACKNOWLEDGEMENTS

Iwoul

d l

i

ketothank my majorprofessorDr.Yong

Chan Lee forhi

s encouragement,advi

ce and gui

dance

throughout thi

s work.Iam gratefulfor Dr.Hoguen

Ki

m and Dr. Woo Ji

n Hyung for servi

ng on my

commi

ttee.Compl

eti

on ofthi

s work has been possi

bl

e

becauseofthei

rreadi

nesstoassi

stmei

nspi

teofthei

r

busyschedul

es.

Iwoul

dl

i

ketothank speci

al

l

y toDr.Ji

e-hyun Ki

m

for her si

ncere advi

ce, suggesti

on, hel

p duri

ng the

courseofresearch and advi

ce and revi

si

on on wri

ti

ng

thi

sthesi

s.Iwanttoextendmy appreci

ati

ontoal

lmy

col

l

eagues,InOhkLee,EunjuRyu,Sang HunLeeand,

Dr.Kyung Mi

n Yang forthei

rhel

p i

n the l

ab work

and fri

endshi

p and express my appreci

ati

on to al

lof

myfri

ends,Myung-EunLee,SookInChung,HyoJung

Shi

m,In HyeShi

m,Mi

yeong Ki

m,Mi

n JiAn and,Se

Ji

n Park for shari

ng joys and sorrows wi

th me

throughoutmyschooldays.

Iam mostthankfuland appreci

ati

veofthesupport

ofmy parents,brotherandsi

ster,whohaveaconstant

sourceofsupportandi

nspi

rati

onal

lthroughmyl

i

fe.

(5)

i

-Table of contents

Abstract ...1

I. Introduction...3

II. Materials and Methods...6

1. Cell and H. pylori culture and infection...6

2. Reagents...6

3. Tissue samples and Immunohistochemistry...7

4. Immunoblotting...8

5. Reverse PCR and real-time reverse PCR analyses...8

6. Statistical analysis...9

Ⅲ. RESULTS...10

1. Shh expression correlated with H. pylori infection in gastric cancer cells...10

2. Overexpression of Shh induced by H. pylori led to enhanced Hh pathway activation in gastric cancer cell...13

3. H. pylori-induced Shh correlated with a reduction in the level of Ptc1 protein...16

4. H. pylori induce Shh protein is related to NF-κB pathway...18

5. Shh expression correlated positively with H. pylori infection in specimens of human gastric cancers...20

Ⅳ. DISCUSSION...24

Ⅴ. REFERENCES...29

(6)

ii

-LIST OF FIGURES

Figure 1. Shh mRNA correlated positively with H. pylori infection in gastric cancer cells...11

Figure 2. Shh mRNA level positively correlated with

H. pylori infection in gastric cancer cells...12

Figure 3. Immunoblotting of Shh expression in two gastric cancer cell lines...13 Figure 5. Effects of H. pylori infection on Gli1 mRNA expression...14 Figure 6. Effects of H. pylori infection on Gli1 protein expression...15 Figure 7. Inhibition of H. pylori-induced Gli1 protein in AGS cells....15

Figure 8. H. pylori-induced Shh correlated with a reduction in

the level of Ptc1 mRNA...16 Figure 9. Real time RT-PCR mRNA expression of Ptc1...17 Figure 10. Immunoblotting of Ptc1 expression in AGS cells...18 Figure 11. Level of H. pylori-induced Shh protein dose

dependently decreased with NF-κB inhibition in AGS cells..19 Figure 12. Level of H. pylori-induced Shh protein dose

dependently decreased with NF-κB inhibition in AGS cells..19 Figure 13. Expressions of Shh in gastric cancer tissues. Shh protein was detected by immunohistochemistry with anti-Shh in

gastric cancer tissues...21 Figure 14. Expressions of Gli1 in gastric cancer tissues. Gli1

protein was detected by immunohistochemistry with

(7)

iii

-LIST OF TABLES

Table 1. Relationship between Shh expression levels and

H. pylori and differentiation...23

Table 2. Relationship between Gli1 expression levels and

(8)

Abstract

T

Th

h

he

e

ee

e

ef

f

ff

f

fe

e

ec

c

ct

t

to

o

of

f

H

He

e

el

li

l

i

ic

c

co

o

ob

b

ba

ac

a

c

ct

t

te

e

er

r

rp

p

py

y

yl

l

lo

o

or

r

ri

i

in

n

nf

f

fe

e

ec

c

ct

t

ti

i

io

o

on

n

o

on

n

ns

s

so

o

on

n

ni

i

ic

c

ch

h

he

e

ed

d

dg

g

ge

e

eh

h

ho

o

og

g

gs

si

s

i

ig

g

gn

n

na

a

al

l

lp

p

pa

a

at

t

th

h

hw

w

wa

a

ay

y

ys

s

si

i

in

n

g

ga

a

as

s

st

t

tr

ri

r

i

ic

c

ce

e

ep

p

pi

i

it

th

t

h

he

e

el

l

li

i

ia

a

al

l

lc

c

ce

e

el

l

ll

l

ls

s

YeunJungChoi

DepartmentofMedicalScience

TheGraduateSchool,YonseiUniversity

(DirectedbyProfessorYongChanLee)

Aberrant activation of hedgehog (Hh) signaling pathway leads to pathologicalconsequencesinavarietyofhumantumors.Sonichedgehog (Shh)isimplicatedinstem/progenitorcellrestitutionofdamagedgastric mucosaduringchronicinfectionwithHelicobacterpylori(H.pylori).H. pyloriwith variousvirulencefactorscausemucosaldamageand may participate in gastric carcinogenesis.The aim ofthis study was to examinethechangesinShhsignalpathwaysingastricepithelialcellsin

(9)

responsetoH.pyloriinfection.WealsoassessedtheinfluenceofcagA andcagE genesofH.pyloriontheShhexpressionbygastricepithelial cells.RealtimeRT-PCR,immunoblotand immunohistochemicalstains wereperformedtoanalyzetheexpression,localizationandtranscriptional regulation of Shh,Patched1 (Ptc1) and Glioma-associated oncogene (Gli1).H.pyloristrains60190(cagPAIpositive),8822(cagPAInegative), andgeneknock-outmutantsH.pyloristrainsofcagA andcagE were used.AGS and MKN45cellswereused asforgastricepithelialcells. Paraffin-embedded tissues from resected human gastric cancers were used to evaluatetheexpression ofShh and related signalproteinsin vivo.Overexpression ofShh in gastric epithelialcells with H.pylori infection was confirmed by immunoblot and real time RT-PCR. Furthermore,cagA positive strains showed higher level of Shh expression.Gli1 mRNA,thetranscriptionaltargetoftheHh pathway, wereoverexpressedincellsinfectedwithH.pyloricomparetouninfected control, implying thatH.pylori infection induced the Hh pathway activationingastricepithelialcells.TheShhpositiveimmunoexpressionin H. pylori-infected gastric cancer tissue was higher than those of uninfectedgastriccancertissuewhiletheimmunostainingofShhcorrelated withtumordifferentiationapartfrom thestatusofH.pyloriinfection.In conclusion,Shh expression correlates with the status ofH.pylori infection andH.pylorimay activatetheShhsignalpathwaysthrough theup-regulationofShhingastricepithelialcells.

빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲 Keywords:Gastriccancer,Hedgehogsignalpathway,Helicobacterpylori

(10)

T

Th

h

he

e

ee

e

ef

f

ff

f

fe

e

ec

c

ct

t

to

o

of

f

H

He

e

el

li

l

i

ic

c

co

o

ob

b

ba

ac

a

c

ct

t

te

e

er

r

rp

p

py

y

yl

l

lo

o

or

r

ri

i

in

n

nf

f

fe

e

ec

c

ct

t

ti

i

io

o

on

n

o

on

n

ns

s

so

o

on

n

ni

i

ic

c

ch

h

he

e

ed

d

dg

g

ge

e

eh

h

ho

o

og

g

gs

si

s

i

ig

g

gn

n

na

a

al

l

lp

p

pa

a

at

t

th

h

hw

w

wa

a

ay

y

ys

s

si

i

in

n

g

ga

a

as

s

st

t

tr

ri

r

i

ic

c

ce

e

ep

p

pi

i

it

th

t

h

he

e

el

l

li

i

ia

a

al

l

lc

c

ce

e

el

l

ll

l

ls

s

YeunJungChoi

DepartmentofMedicalScience

TheGraduateSchool,YonseiUniversity

(DirectedbyProfessorYongChanLee) I I I...IIINNNTTTRRROOODDDUUUCCCTTTIIIOOONNN

The Hedgehog (Hh)signaling pathway is intimately linked to cell growth and differentiation, with physiological roles in embryonic pattern formation,adulttissue homeostasis and pathologicalroles in tumorinitiationandgrowth.(1)_Intheadul_t,Hhsi_gnal_i_{ng remai}_nsacti_ve

(11)

in some organs where it has been implicated in the regulation of stem-cell maintenance and proliferation.(2) _{The contri}_buti_{on of Hh}

pathway tothedevelopmentand homeostasisofgastricgland hasbeen reported.(3)_Mammal_i_anHhprotei_nsi_ncl_udesoni_cHh(Shh),i_ndi_anHh(Ihh),

anddesertHh (Dhh).Hh ligandsundergopost-translationalmodifications, including autocatalytic cleavage and coupling of cholesterol to the amino-terminalpeptide,which is the fragment that possesses allthe signaling activity.(1) _{In the absence of Hh si}_gnal_i_{ng,patched fami}_l_y

receptors (Ptch1/Ptch and Ptch2)inhibitthe smoothened (Smo)signal transducer.(4)_Hhbi_ndi_{ngtoPtchremovesthei}_nhi_bi_{toryeffectandal}_l_ows

Glito enter the nucleus and act as a transcriptionalactivator.Hh signaling activation leads to Gli-dependenttranscriptionalactivation of targetgenes,suchasGli1,Ptch1,cyclinD2(CCND2),ForkheadBoxM1 (FoxM1)andJagged-2(Jag2).(5)_Hhsi_gnal_i_{ng targetsi}_ncl_udegenesthat

areimportantforcellproliferationandgrowth.Shhisup-regulatedina variety of human tumors,such as gastric cancer,pancreatic cancer, esophagealcancer,and prostatic cancer.Shh up-regulation through the aberrant activation of Hh signaling pathway may leads to carcinogenesis.(6)

Gastric cancer is frequently associated with chronic Helicobacter pylori (H. pylori) infection and is one of the most common malignancies in the world.(7) _Accumul_ati_{on of geneti}_{c al}_terati_{ons or}

epigenetic changes caused by genetic predisposition,life style,and persistentmucosaldamageassociated with chronicH.pyloriinfection may eventually leadsto gastriccancer.(4), (8)_{H.pyloristrainscan be}

dividedintotwomajorsubpopulationesbasedontheirabilitytoproducea 120-145 kDa immunodominant protein, cytotoxin-associated gene A (CagA)antigen.(9)_Cl_i_ni_cal_l_y,i_nfecti_{on wi}_{th the}_cagA-posi_ti_ve_H.pylori

(12)

strain has been associated with higher degree of gastric mucosal inflammationaswellassevereatrophicgastritisandhasbeensuggested toplayanimportantroleinthedevelopmentofgastriccancer.(10)

H.pyloriinduced nuclear factor κB (NF-κB) activation in MKN45 gastric epithelialcells through the ligation oftoll-like receptor(TLR)2 and TLR5,butnotTLR4.(11)NF-κB,a transcriptionalregulator,controls expression of numerous genes involved in inflammation and immune response processes, including proliferation, invasion and adhesion, angiogenesis,and apoptosis.(12) It has also been reported that NF-κB activation up-regulates expression ofShh,resulting in activation ofHh signalingincancer.(13)

Thisstudy examinedthechangesinShhsignalpathwaysingastric epithelialcellsin responsetoH.pyloriinfection.Wecheckedwhether H.pyloriinfection could lead to enhanced Hh pathway activation in gastriccancercellsandthiswasassociatedwithcagA positivestrains. We then examined the role of NF-κB pathway activation on Shh expressioninvariousgastriccancercellsinfectedwithH.pylori.

(13)

I I IIII...MMMAAATTETEERRRIIIAAALLLSSS AAANNNDDD MMMEEETTTHHHOOODDDSSS 1 1 1...CCCeeellllllaaannndddH.HH..pppyyylllooorrriiicccuuullltttuuurrreeaeaannndddiiinnnfffeeecccttitiiooonnn...

AGS (ATCC CRL 1739) and MKN45 (KCLB No.80103) gactric epithelialcells,weremaintained in RPMI1640 medium (Gibco,Grand Island.NY)supplementedwith10% fetalbobineserum (Gibco)and1% penicillin-streptomcin sulfate (Gibco).Allcultures were maintained at 37°C incubator supplemented with 5% CO2.H.pyloristrains were

cultured on blood agar plate containing 5% sheep blood at 37°C incubatorwith gas pak (BD,Sparks,MA).We cultured 8822 (PAI-negative strain) and 60190 (PAI-positive strain) along with △cag (cagA knock-out isogenic mutant) strains(14) _(ki_{nd gi}_{ft from Prof.}

Richard M.Peek.Jr.,VanderbitUniv.).ForH.pyloriinfection with gastric epithelialcells,H.pyloristrains were cultured for24-36 hr. CulturedH.pyloriwereharvested in PBS (pH 7.4)and added tothe serum-starvedhostcellsatamultiplicityofinfection(M.O.I)of50. 2

2

2...RRReeeaaagggeeennntttsss

The Smo-specific inhibitorcyclopamine (Calbiochem,La Jolla,CA) wasdissolvedinDMSO. ProteasomeinhibitorMG-132(Sigma-Aldrich, St.Louis,MO)andpyrrolidinedithiocarbamate(PDTC)(Sigma-Aldrich), aspecificinhibitorofNF-κB,wereusedattheconcentrationspreviously described.(15)

(14)

3 3

3...TTTiiissssssuuueeesssaaammmpppllleeessasaannndddIIImmmmmmuuunnnooohhihiissstttooocchchheeemmmiiistssttrrryyy...

Surgical specimens were obtained from 20 patients with gastric cancer.Tissuesectionsin microslidesweredeparaffinizedwith xylene, hydrated in serialdilutions ofalcohol,and immersed in 3% H2O2 to

quench endogenous peroxidase activity.Following antigen retrievalin citratebuffer(pH 6.0),thetissuesectionswereincubated with protein blockingagent(Immunotech,Coulter,Inc.,Marseille)toblocknonspecific antibody binding for20 min atroom temperature and then incubated overnightat4°C with an optimaldilution ofa primary polyclonalgoat antibody againsthuman Shh (H-160,Santa Cruz),Ptc1 (G-19,Santa Cruz),Gli1 (H-300,Santa Cruz).Afterwashing with PBS threetimes, the sections were incubated with the biotinylated secondary antibody (goat anti-rabbit IgG, Immunotech, Coulter, Inc., Marseille) and streptavidin conjugated to horseradish peroxidase (Immunotech)for 20 min atroom temperature,followed by PBS wash.Thechromogen was developed for 5 min with liquid 3,3′-diaminobenzidine (Immunotech). Next, slides were counterstained with Meyer's hematoxylin, then dehydrated,andmountedwithhistochoicemountingmediaforexamination. A total of 100-150 cancer cells were counted in each section.For immunostaining ofShh,cancercells showing no detectable cytoplasm staining were given뺳negative뺴and strong cytoplasm staining were given뺳positive'.Forimmunostaining ofGli1,cancercellsshowing no detectable nuclear staining were given뺳negative뺴and strong nuclear stainingweregiven 뺳positive'.

(15)

4 4

4...IIImmmmmmuuunnnooobblblloootttttitiinnnggg...

Preparedcellswereharvestedafterwashing withphosphatebuffered saline.Collectedcellswerelysedwithbuffer(50mM Tris-Cl(pH 7.5), 150mM NaCl,1mM EDTA (pH 8.0),1% TritonX-100,1mM PMSF, 1mM Na3VO4,proteaseinhibitorcocktail(Roche,Molecularbiochemical

Indianapolis,IN)).Same amountofprotein was boiled at95°C after adding SDS sample buffer (62.5 mM Tris-Cl(pH 6.8),2% sodium dodecylsulfate,10% glycerol,β-mercaptoethanol,0.002% bromophenol blue).Sampleswereloaded in 12% SDS-PAGE forShh and 8% SDS PAGE for Ptc1 and Gli1 and transferred to PVDF membrane (Amersham Biosciences,Pisctaway NJ).Rabbitanti-Shh,anti-Gli1and goatanti-Ptc1(SantaCruz)wereusedastheprimarylabelingantibodies and theappropriatehorseradish peroxidase–conjugated antibodies(Santa Cruz)wereusedassecondaryantibodies.Anenhancedchemiluminescence detection system (ECL-Plus, iNtRON, Seoul, Korea) was used for detection. 5 5 5... RRReeevvveeerrrssseee tttrrraanannssscccrrriiippptttiiiooonnn---PPPCCCRRR aaannnddd rrreeeaaalll---tttiiimmmeee rrreeevvveeerrrssseee t t trrraaannnssscccrririippptttiiiooonnn---PPPCCCRRR aaannnaalallyyyssseeesss...

TotalRNA wasextractedfrom culturedcellsusingaRNeasyminikit (Qiagen,Tokyo,Japan).The RNA wasreversetranscribedusing oligo (dT)12-18 primers and superscripTM Ⅱ reverse transcriptase (Invitrogen,

Carlsbad,CA).PCR wasdonewith PCR Maxikit(iNtRON,Sungnam, Korea)according to the manufacturer's instructions.The amplifications weredoneby denaturation at95°C for5min,followedby 30cyclesof 30seceachat95°C,57°C,and72°C forShhandPtc1,30cyclesof30

(16)

seceachat95°C,59°C,and72°C forGli1and18cyclesof30seceach at95°C,60°C,and 72°C for β-actin.Thefollowing primerpairswere used: β-actin,5'-TTG CCG ACA GGA TGC AGA AGA-3' and 5'-AGG TGG ACA GCG AGG CCA GGA T-3';Shh,5'-GAG ATG TCT GCT GCT AGT CC-3'and5'-GTT TCT GGA GAT CTT CCC TT-3';Ptc1,5'-ATG CTG GCG GGA TCT GAG TTC GAC T-3' and 5'-GGG TGT GGG CAG GCG GTT CAA G-3';Gli1 5'-TAT GGA CTT CCC ACC TAC TG-3'and5'-AAT GTT CAA GTC GAG GAC AC-3'.

The real time RT-PCR analysis was carried out with a PCR mixturecontaining 1µmol/L ofeach primerand SYBR Green master mix (Applied Biosystems,FosterCity,CA).The amplifications were conducted at95°C for 10 sec and 60°C for 60 sec using the ABI PRISM 7000 Quantitative PCR system (Applied Biosystems(16)_).Each

sample was examined in triplicate and the amounts of the PCR products produced were normalized with respect to β-actin as an internalcontrol.

6 6

6...SSStttaaatttiiissstttiiicaccaalllaaannnaaalllyyysssiiisss

Statisticalanalyses were conducted using Studentt-test for real time PCR and Pearson's chi-square test for immunohistochemistry. p-values< 0.05wereregardedasstatisticallysignificant.Allstatistical analyseswereperformedusingSPSS software(SPSS 12.0,Chicago).

(17)

ⅢⅢⅢ...RRREEESSSUUULLLTTTSSS

1 1

1...SSShhhhhh eeexxxpprprreeessssssiiiooonnn ccocoorrrrrreeelllataatteeeddd wwwiiitththhHHH...pppyyylllooorrriiiiiinnnfffeeecctcttiiiooonnn iiinn gnggaaassstttrrriiiccc cccaaannnccceeerrrccceeellllllsss...

To examine whether H. pylori infection was associated with expressionofShhmRNA andprotein,weperformedinvitroexperiments withthreehumangastriccancercelllines,AGS andMKN74.Wefirstly examinedwhetherH.pyloriinfectionalteredShhexpressioninthesecell lines.Weinfectedthesecelllineswith8822(PAI-negativestrain),60190 (PAI-positive strain)and isogenic mutants △cagA for6 or24 hrto induceShhexpression.TheexpressionofShhmRNA wasexaminedby real-timereversetranscription PCR orRT-PCR in gastriccancercells. On the whole,H.pylori infection increased the expression of Shh mRNA,and 60190 infection enhanced the expression Shh mRNA more significantlythan8822and△cagA (Fig.1,2).

Toconfirm thattheShh production relatedtoH.pyloriinfection in gastric cancercells,we performed immunoblotting analysis.H.pylori infection increased Shh levels in AGS and MKN45 cells (Fig.3). Furthermore,infectionbycagA positivestrainsshowedhigherlevelof Shh expression when infected (Fig.3).These findings indicated that H.pyloriinfection affected Shh mRNA expression and enhanced Shh proteinproductioninthesecelllines.

(18)

Fig.1.Shh mRNA correlated positively withH.pyloriinfection in gastric cancercells.AGS and MKN45 cells were infected byH.pyloriCagA positive (60190) and negative (△cagA, 8822) strains for 6 hr and RT-PCR were performed.β-actin served as an internalcontrol.60190 significantlyenhancedexpressionShhmRNA.

(19)

Fig.2.Shh mRNA levelpositively correlated withH.pyloriinfection in gastric cancer cells. Shh mRNA expressions by AGS cells were examined by real-time RT-PCR. Relative Shh mRNA level after normalization to the corresponding β-actin mRNA expression were shown.H.pyloriincreasedShh mRNA levelsin AGS cell.Furthermore,

cagA positive strains showed greater levelofShh mRNA expression when infected.Columns,mean of the three independent experiments;

(20)

Fig.3.Immunoblotting ofShh expression in two gastric cancercelllines. AGS andMKN45cellswereinfectedwith8822,60190and△cagA for6 or 24 hr to induce Shh expression.H. pyloriinfection increased expression of Shh protein, and 60190 other than 8822 and △cagA significantly increased the expression ofShh protein in gastric cancer cellswheninfected.

2 2

2...OOOvvveeerrreexexxppprrreeessssssiiiooonnn ooofffSSShhhhhh iiinnnddduuucceceeddd bbbyyyHHH..p.ppyyylllooorrriiillleeeddd tttooo eeennnhhhaaannnccceeeddd HHHhhhpppaaattthhhwwwaaayyyaacacctttiiivvvaaatttiiiooonnniiinnngggaaassstttrrriiiccccccaaannnccceeerrrccceeellllll...

We examined the ligand-dependentHh pathway activation in gastric cancercells.The gastric cancercells were infected withH.pylorito overexpressShh,and theGli1expression,atranscriptionaltargetofthe Hh pathway,was then monitored by RT-PCR and immunoblotting.(16)

RT-PCR revealedthatcellsinfectedwithH.pyloriexpressedhigherlevel ofGli1mRNA thanuninfectedcontrol(Fig.5)andImmunoblottingshowed that60190strainsignificantlyinducedgreatermagnitudeofGli1proteinin AGScells(Fig.6).

(21)

Fig.5.Effects ofH.pyloriinfection on Gli1 mRNA expression.AGS and MKN45cellswereinfectedwithH.pylorifor6hr.RT-PCR analysisof theexpression ofGli1 mRNA wereperformed.H.pyloriincreased Gli1 mRNA levelsin thesecells.Furthermore,cagA positivestrainsshowed higherlevelofGli1mRNA expression when infected.H.pyloriinfection inducedGli1mRNA expressionimplyingenhancedHhpathwayactivation ingastriccancercells.

These data suggested that overexpression of Shh enhanced Hh pathwayactivationwithinthegastriccancercells.

To confirm theseresults,wetreated AGS cellwith cyclopamineto suppress Smo (Fig. 7). Immunoblotting revealed that cyclopamine significantly suppressed theexpression ofGli1protein (Fig.7).These data again confirmed thatH.pylori-induced Shh expression activated theHhpathwayingastriccancercells.

(22)

Fig.6.Effects ofH.pyloriinfection on Gli1 protein expression.AGS cells wereinfectedwith8822, 60190and△cagA for6or24hrtoinduceGli1 expression. Gli1 protein by immunoblotting in AGS cells. 60190 significantly induced greateamountofGli1protein expression compareto 8822 and △cagA.H. pyloriinfection induced Gli1 protein implying enhancedHhpathwayactivationingastriccancercells.

Fig.7. Inhibition ofH.pylori-inducedGli1proteinin AGS cells.AGS cells were infected withH.pylorifor12 hrafterthey were pretreated with cyclopamine for 6 hr. Immunoblotting was done with anti-Gli1. CyclopaminedosedependentlyinhibittheexpressionofGli1proteingastric cancercellswheninfectedbyH.pylori.

(23)

3 3

3...HHH...pppyyylllooorrriii---iiinnnddduuuccceeedddSSShhhhhhccocoorrrrrreeelllaaattteeedddwwwiiittthhhaaarrereeddduuuccctttiiionoonniiinnnttthhheeellleeevvveeelllooofff PPPtttccc111ppprrrooottteeeiiinnn...

We examined whether Hh signaling has the paradoxicaleffectof both blocking Ptcprotein function and activatingptcgeneexpression. We, unexpectedly observed thatH. pylori infection had opposing effectsonPtc1.Ptc1expressionwasreducedinAGS cellinfectedwith H.pyloriand 60190 strain was sufficientto reduce Ptc1 expression (Fig.8-10).These observations suggestthatH.pylori-induced Shh causeddownregulationofPtc1proteiningastriccancercells.

Fig.8.H.pylori-inducedShhcorrelatedwithareductioninthelevelofPtc1 mRNA.AGS cellswereinfectedbyH.pyloriCagA positive(60190)and negative(△cag,8822)strainsfor6hrand RT-PCR wereperformed.β -actin served as an internal control.H. pylori starin of 60190 significantly reduced expression Ptc1mRNA.Ptc1mRNA reduction was dependentonCagA proteiningastriccancercells.

(24)

Fig.9.Realtime RT-PCR mRNA expression of Ptc1.AGS cells were infected byH.pyloriCagA positive (60190)and negative (△cag,8822) strains for6 hrand Realtime RT-PCR were performed.Relative Ptc1 mRNA levelafter normalization to the corresponding β-actin mRNA expressionwereshown.H.pylori(cagApositivestrain)infectionreduced Ptc1mRNA levelsin AGS cell.Columns,mean ofthethreeindependent experiments;bars,SD.

(25)

Fig.10.Immunoblotting ofPtc1 expression in AGS cells.AGS cells were infected by H.pyloristrains for 8 hr and 24 hr and analyzed by immunoblotting with anti-Ptc1.H.pylorireduced expression of Ptc1 protein and moreover 60190 significantly reduced expression of Ptc1 proteiningastriccancercells.HeLacellisapositivecontrol.

4 4

4...HHH...pppyyylllooorrriiiiiinnnddduuuccceeeSSShhhhhphpprrrooottteeeiiinnniiisssrrreeelllaaattteeedddtttoooNNNFFF---κκκκBBB pppaaattthhhwwwaaayyy... H.pylorihas been demonstrated to induce chemokine and NF-κB activation through TLR2 and TLR5.(12) _{We questi}_{oned whether} _H.

pylori-inducedoverexpressioninShhproteinwouldbeblockedbyNF-κB inhibition.AGS cellswerepretreated with theNF-κB inhibitor,MG-132 (Sigma-Aldrich,St.Louis,MO),before infection withH.pylorifor6 hours.The expression ofShh protein was analyzed by immunoblotting (Fig.11).MG-132treatmentresultedinanearcompleteinhibitionofShh protein expression.These results indicate thatH.pylori-induced Shh expressioninAGScellsismediatedthroughNF-κB activation.Toconfirm theseresults,AGS cellsweretreatedwithH.pylorifor6hrafterthey were pretreated with another specific NF-κB inhibitor, pyrrolidine dithiocarbamate(PDTC)for6hr(Fig.12).(17)_{Whentreatedwi}_thH.pylori

in the presence of PDTC,levels of Shh were reduced.These data suggestedthattheH.pylori-inducedShhproteinexpressionwasmediated throughNF-κB pathwayingastriccancercells.

(26)

Fig.11.LevelofH.pylori-induced Shh protein dosedependently decreased with NF-κB inhibition in AGS cells.AGS cells were infected withH. pylorifor 12 hr after they were pretreated with MG-132 for 1 hr. Immunoblotting was done with anti-Shh.When the concentration of MG-132washigherthan2 μM MG-132couldinhibit expressionofShh proteininducedbyH.pylori.

Fig.12.LevelofH.pylori-induced Shh protein dosedependently decreased with NF-κB inhibition in AGS cells.AGS cells were infected withH. pylorifor 6 hr after they were pretreated with PDTC for 6 hr. Immunoblotting was done with anti-Shh.When the concentration of PDTC was higherthan 50 μM PDTC could inhibit expression ofShh proteininducedbyH.pylori.

(27)

5 5 5...SSShhhhhh eeexxxppprrreeessssssiiiooonnn ccocoorrrrrreeelllaaatteteeddd pppooosssiiitttiiivveveelllyyy wwwiiithtthhHHH...pppyyylllooorrriiiiiinnnfffeeecctcttiiiooonnn iiinnn s s spppeeeccciiimmmeeennsnssooofffhhhuuummmaaanngnggaaassstttrrriiiccccccaaannnccceeerrrsss...

To examine whether H. pylori infection was associated with expression of Hh pathway in vivo, we stained a series of 20 paraffin-embedded surgicalspecimens,including 10 specimens ofwell differentiated carcinomas (H.pylori-positive group and negative group) and10specimensofpoorly differentiatedcarcinomas(H.pylori-positive groupandnegativegroup).ExpressionsofShhandGli1wereexamined immunohistochemically.Shhligandwasexpressedmainlyinthecytoplasm (Fig.13)and Gli1 was strongly expressed in both the cytoplasm and nucleiofgastriccancercells(Fig.14).H.pylori-positivegroup showed higherShhpositivestainingratiosthanH.pylori-negative(Table1.).The percentage ofcells with nuclearstaining ofGli1 showed similarresults (Table2.).Inaddition,thestainingratiosofwelldifferentiatedcarcinomas werehigherthanthoseofpoorlydifferentiatedcancerandthisassociation wasstatistically significant(18)_{.A strong posi}_ti_vecorrel_ati_{on wasdetected}

between Shh staining ratioandGli1.Thesedatasuggestedthat1)there wasacorrelationbetweenH.pyloriinfectionandShhexpression,and2) overexpressionofShhinducedbyH.pyloriinfectionmayinduceenhanced Hhpathwayactivationingastriccancertissues.

(28)

Fig.13.ExpressionsofShhingastriccancertissues.Shhproteinwasdetectedby immunohistochemistrywithanti-Shhingastriccancertissues.Shhligandwas expressed mainlyinthecytoplasm ofgastriccancercells.H.pylori-negative tissue was less intense than H.pylori-positive.Shh expression in well differentiated cancerwas strongerthan thatin poorly differentiated cancer. Magnification:×400

WD Hp+:WelldifferentiatedandH.pyloripositive. WD Hp-:WelldifferentiatedandH.pylorinegative. PD Hp+:PoorlydifferentiatedandH.pyloripositive. PD Hp-:PoorlydifferentiatedandH.pylorinegative.

(29)

Fig.14.Expressions of Gli1 in gastric cancer tissues.Gli1 protein was detected by immunohistochemistry with anti-Gli1 in gastric cancertissues. Gli1 was strongly expressed in both the cytoplasm and nucleiofgastric cancer.H.pylori-negativetissuewaslessintensethanH.pylori-positive. Gli1 expression in welldifferentiated cancer was stronger than that in poorlydifferentiatedcarcinomas.Magnification:× 400

WD Hp+:WelldifferentiatedandH.pyloripositive. WD Hp-:WelldifferentiatedandH.pylorinegative. PD Hp+:PoorlydifferentiatedandH.pyloripositive. PD Hp-:poorlydifferentiatedandH.pylorinegative.

(30)

Table 1.Relationship between Shh expression levels and H.pyloriand differentiation.

ForimmunostainingofShh,cancercellsshowingnodetectablecytoplasm staining weregiven뺳negative뺴andstrongcytoplasm stainingweregiven뺳positive'.

p< 0.05,p-valuescalculatedusing:chi-squareanalysis. WD:welldifferentiated +:H.pyloripositive

PD:poorlydifferentiated. -:H.pylorinegative

Table 2. Relationship between Gli1 expression levels and H.pyloriand differentiation.

Forimmunostaining ofGli1,cancercellsshowing nodetectablenuclearstaining weregiven뺳negative뺴andstrongnuclearstainingweregiven 뺳positive'.

p< 0.05,p-valuescalculatedusing:chi-squareanalysis. WD:welldifferentiated +:H.pyloripositive PD:poorlydifferentiated. -:H.pylorinegative

(31)

Ⅳ Ⅳ

Ⅳ...DDDIIISSSCCCUUUSSSSSSIIIOOONNN

It was recently reported that aberrant activation of Hedgehog signaling pathway lead to pathologicalconsequencesin human gastric cancer,pancreaticcancer,esophagealcancer,prostatecancer,smallcell lung cancer,basalcellcarcinoma,medulloblastoma and glioma.(6, 19,

20-22)_{TheShh-Smo-Gl}_i_{1pathway hasapi}_votalrol_ei_{n thegrowth of}

gastriccancercellsand thestimulation ofHh signaling by Shh ligand occurs in an autocrine/paracrine manner in gastric cancer cells.(16) However,themolecularmechanismsunderlying thisabnormalactivation remain unclear.Cyclopamine,a steroidalalkaloid thatinteracts directly with Smo to inhibits Hh signaling,effectively retards the growth of various tumors, including gastric carcinoma, indicating that Hh signaling isinvolvedin tumorgrowth.(19, 23-25)_Neverthel_ess,i_tremai_ns

unclear how the blockade of Hh signaling leads to tumor growth suppression in gastric cancer.Ifso,itis importantto elucidate the identities ofthe molecules thatregulate expression ofShh.Ourdata thatH.pyloriinfection isoneofthemechanismsresponsibleforShh overexpressionseem tohavebroadmeaning.

H.pylori primarily infects individuals during childhood,and can cause gastric diseases such as chronic atrophic gastritis and peptic ulcerslaterinlife.(8)_Recentepi_demi_ol_ogi_calstudi_eshavei_ndi_catedthat

H.pyloriisinvolvedinthedevelopmentofgastricadenocarcinoma.A large-scaleprospectivestudy revealed thattherisk fordevelopmentof gastriccarcinomawasmuchgreaterintheH.pylori-infectedpopulation than in the H. pylori-uninfected population.(26) However, molecular mechanismsby whichH.pyloritriggerstheprocessleading togastric carcinomaremainlargelyunknown.

(32)

Wefocused on theroleofH.pyloriinfection in Shh expression in gastric cancer for the following reason.Chronic persistent infection withH.pylorioverafew decadesleadstochronicatrophicgastritis, and then to gastric cancer.(7)_{H.pyloriwith CagA virulence factors}

causes mucosaldamage,and following repair in the stomach.Shh, ensuring stem/progenitorcellrestitutionofdamagedgastricmucosa,is implicated in the repair process during chronic persistentH.pylori infection.Ourdata showed thatCagA positivestrainsshowed higher levelofShhexpression when infected.Thevastmajority ofH.pylori strains contain the cag pathogenicity island (cag-PAI), a 40 kb genomicfragmentcontaining 31genes.ThisstretchofDNA codesfor type IV secretion (TFSS)apparatus used to injectbacterialproteins suchasthe120kD proteinCagA intohostepithelialcells.A seriesof in vitroreportshavenow establishedthatinjection ofCagA intohost cells leads to phosphorylation ofCagA by hostcellkinases (such as c-Src),resulting in activation ofSHP-2tyrosinephosphataseandMAP kinase signaling pathways.(27) _{CagA i}_{s an i}_{mportant medi}_{ator for}

cagPAI-dependentinduction ofpotentproinflammatory responsesduring H.pyloriinfection.TransfectedandtranslocatedCagA from asubsetof H.pylori strains are able to induce IL-8 release through NF-κB activation.(28)

NF-κB isatranscriptionfactorthatisinvolvedininflammation,(29)_cel_l

proliferation,(30)_angi_ogenesi_s,(31)_{and apoptosi}_s.(32)_{NF-κB i}_{s consti}_tuti_vel_y

activated in gastric carcinoma and NF-κB activation is related to the invasiveability ofcarcinomacells.(33)_{In mostunsti}_mul_{ated,normalcel}_l_s,

NF-κB ispresentinthecytoplasm asaninactiveheterodimercomposed ofthe p50,p65,and IκBα subunits.After activation,IκBα undergoes phosphorylation and ubiquitination dependent degradation by the

(33)

proteasome.Consequently,nuclear localization signals on the p50-p65 heterodimerareexposed,leading tonucleartranslocationandbinding toa specific consensus sequence that activates gene transcription,including genes encoding inflammatory cytokines,chemokines,growth factors,cell adhesion molecules,andcytokinereceptors.(34)_{NF-κB acti}_vati_{on i}_soneof

the mechanisms underlying Shh overexpression in pancreatic cancerand that proliferation of pancreatic cancer cells is accelerated by NF-κB activation in partthrough Shh overexpression.(13)_Toexami_newhetherH.

pyloriinduceShh protein via activation ofNF-κB,weused proteasome inhibitorMG-132andPDTC.PDTC isaspecificinhibitorofNF-κB and belongs to an antioxidant.Itfunctions through two pathways,one is inhibiting NF-κB p65 protein activation,another is reducing NF-κB nucleitranslocationbyinhibiting degradationofκB inhibitorymolecule. When the concentration ofMG-132 was higherthan 2 μM,MG-132 couldinhibit expressionofShhproteininducedbyH.pylori.and50 μ M PDTC could inhibit expression ofShh protein via activation of NF-κB.Togetherwith these results,ourdata raised a possibility that CagA inducedShhproteinthroughNF-κB activation.

TheHh signaling pathway isataleoftwotransmembraneproteins Patched (Ptc1), a twelve-pass membrane protein binds Hedgehog ligand.Smoothened(Smo),aseven-passmembraneprotein isasignal transducer.Ithadbeen shown thatShh correlatedwithareduction in thelevelofPtc1protein.In theabsenceofligand,Ptcinteractswith and inhibits Smo, either directly or indirectly. This repression culminates in a transcription factor acting as a transcriptional repressor.Thetranscription factoriscalledCubitusinterruptus(Ci)in Drosophilaand Gliin vertebrates.CellsthatreceivetheHh signal,in contrast,expressthesegenesbecausePtccannotfunction.Therelative

(34)

levelsofopposing Hh and Ptcactivitiesthuscontrolwhereandwhen targetgenesareexpressed.Interestingly,ptcitselfisnearlyalwaysinduced byHh.(35)_{Therefore,Hhsi}_gnal_i_{nghastheparadoxi}_{caleffectofbothbl}_ocki_ng

Ptcprotein function andactivatingptcgeneexpression.Shh elevatesptc1 expressioninmosttissues,confirmingthatptc1isanexcellentindicatorof Hhsignaling.(36)Conversely,persistentandhigh-levelPtcexpressionfrom a transgene expressed throughout the neuraltube inhibits transcription of endogenousptc1.(37)_{Ourdata showed thatPtc1 protei}_{n and mRNA were}

reduced in AGS cellinfected with 60190.Thedetailed mechanisms that down-regulation ofPtc expression byH.pyloriinfection remains to be investigated.

In the present study,we showed that theexpression of Shh in gastricepithelialcellsinfectedbyH.pyloriwasincreasedwhichwere confirmedby immunoblotandrealtimeRT-PCR.Also,CagA positive strains showed higherlevelofShh expression byH.pyloriinfected gastric cancer cells. Gli1 mRNA and protein were increased in expression in cells infected withH.pyloriimplying the activation of Shh signal pathway in gastric cancer cells.H. pylori infection contributes to Hh pathway activation through up-regulation of Shh expression in gastric cancer cells. We demonstrated a positive correlation betweenH.pyloriinfection and Shh expression in clinical tissuesamples.CellsinfectedwithH.pyloriexpressedhigherlevelsof Shh and Gli1than theuninfected controlcells.Thesefinding indicate that H. pylori-induced overexpression of Shh activated the Hh pathway in gastric cancer cells in a ligand-dependentmanner.The reduction in Shh protein expression by inhibitors of NF-ĸB signal pathway suggest the possible role of NF-ĸB pathway in Shh expressionbyH.pyloriinfectedgastriccancer.

(35)

Inconclusion,ShhexpressioncorrelateswiththestatusofH.pylori infection andH.pylorimay activatetheHh signalpathwaysthrough up-regulationofShhingastricepithelialcells.CagA mayinfluencethe expressionofShhingastricepithelialcells.Thisnovelfinding thatH. pyloriinfection influencesShh expression improvesourunderstanding ofthemechanism ofHhsignalingactivationingastriccancer.

(36)

Ⅴ Ⅴ

Ⅴ...RRREEEFFFEEERRREEENNNCCCEEESSS

1.Ingham PW,McMahon AP:Hedgehog signaling in animaldevelopment: Paradigmsandprinciples.GenesDev2001;15:3059-87.

2.Pasca diMagliano M and Hebrok M:Hedgehog signaling in cancer formationandmaintenace.NatRevCancer2003;3:903-11.

3.Ramalho-Santos M,Melton DA,McMahon AP:Hedgehog signals regulate multipleaspectsofgastrointesinaldevelopment.Development2000;127:2763-72. 4.KatohY andKatohM:Hedgehog signaling ingastriccancer.CancerBiol

Ther2005;4:1050-54.

5.CarpenterD,StoneDM,BrushJ,etal.:Activating Smoothenedmutations insporadicbasal-cellcarcinoma.Nature1998;391:90-92.

6.Berman DM,Karhadkar SS,Maitra A,Montes OR,Gerstenblith MR, BriggsK,ParkerAR,ShimadaY,Eshleman JR,WatkinsDN,Beachy PA: Widespread requirement for Hedgehog ligand stimulation in growth of digestivetracttumors.Nature2003;425:846-9.

7.Katoh M,TeradaM,NishiM:Oncogenesandtumorsuppressorgenesin Gastriccancer.Tokyo:Splinger-Verlag,1993;196-208.

8.Hatakeyama M:Oncogenic Mechanisms ofthehelicobacter pyloriCagA protein,Nat.Rev.Cancer2004;4:688-94.

9.CovacciA,CensiniS,BugnoliM etal:Molecularcharacterization ofthe 120-kDa immunodominant antigen ofHelicobacter pyloriassociated with cytotoxicityandduodenalulcer.ProcNatlAcadSciUSA 1993;90:5791-5. 10.KuipersEJ,Perez-PerezGI,MeuwissenSG,BlaserMJ:Helicobaterpylori

nd atrophic gastritis:importance ofthecagA status.J NatlCancer Inst 1995;87:1777-80.

11.Smith MF,Jr.,Novotny J,CarlVS,Comeau LD:Helicobacterpyloriand toll-like receptor agonists induce syndecan-4 expression in an NF-κ B-dependentmanner.Glycobiology2006;3:221-29.

12.KarinM,GretenFR:NF-κB:linkinginflammationandimmunitytocancer developmentandprogression.NatRevImmunol.2005;5:749-59.

(37)

13.Nakashima H,Nakamura M,YamaguchiH,Yamanaka N,AkiyoshiT, Koga K et al.: Nuclear Factor-κB Contributes to Hedgehog Signaling Pathway ActivationthroughSonichedgehog Induction inPancreaticCancer. CancerRes2006;66:7041-49.

14.HigashiH,Nakaya A,TsutsumiR,Yokoyama K,FujiiY,Ishikawa S, HiguchiM,TakahashiA,KurashimaY,TeishikataY,TanakaS,AzumaT, Hatakevama M: Helicobacter pylori CagA induces Ras-independent morphogenetic response through SHP-2 recruitmentand activation.J Biol Chem,2004;279:17205-10.

15.LiQ,YuYY,ZhuZG,JiYB,ZhangY,LiuBY,ChenXH,LinYZ:Effect of NF-kappaB Constitutive Activation on Proliferation and Apoptosis of GastricCancerCellLines.EurSurgRes2005;37:105-10.

16.OhtaM,TateishiK,KanaiF,etal.:p53-Independentnegativeregulation of p21/cyclin-dependent kinase interacting protein 1 by the sonic hedgehog-glioma-associated oncogene1pathway in gastriccarcinomacells. CancerRes2005;65:10822-29.

17.Ragione FD,Cucciolla V,Borriello A,Pietra VD,Manna C,GallettiP, Zappia V:Pyrrolidine dithiocarbamate induces apoptosis by a cytochrome c-dependentmechanism.Biochem BiophysResCommun2000;268:942-46. 18.AkiyoshiT,NakamuraM,NakashimaH,Yao T,TsuneyoshiM,Tanaka

M,KatanoM:Gli1,downregulatedincolorectalcancers,inhibitsproliferation ofcoloncancercellsinvolvingWntsignalingactivation.Gut2006;55:991-99. 19.ThayerSP,Magliano MP,HeiserPW,etal.:Hedgehog is an early and

latemediatorofpancreaticcancertumorigenesis.Nature2003;425:851-56. 20.Johnson RL,Rothman AL,Xie J,Goodrich LV,Bare JW,Bonifas JM,

Quinn AG,MyersRM,Cox DR,Epstein JrEH,ScottMP:Human homolog ofpatched,a candidate gene for the basalcellnevus syndrome.Science 1996;272:1668-71.

21.XieJ,MuroneM,Luoh SM,Ryan A,Gu Q,Zhang C,BonifasJM,Lam CW,Hynes M,Goddard A,RosenthalA,Epstein JrEH,de Sauvage FJ: Activating Smoothened mutations in sporadic basal-cellcarcinoma.Nature

(38)

1998;391:90-92.

22.RaffelC,Jenkins RB,Frederick L,Hebrink D,Alderete B,Fults DW, JamesCD:Sporadicmedulloblastomascontain PTCH mutations.CancerRes 1997;57:842-45.

23.Watkins DN,Berman DM,BurkholderSG,Wang B,Beachy PA,Baylin SB: Hedgehog signalling within airway epithelial progenitors and in small-celllungcancer.Nature2003;422:313-17.

24.SanchezP,HernandezAM,Stecca B,KahlerAJ,DeGuemeAM,Barrett A,Beyna M,Datta MW,Datta S,RuizI,Altaba A:Inhibition ofprostate cancer proliferation by interference with Sonic Hedgehog-GLI1 signaling. ProcNatlAcadSciUSA 2004;101:12561-66.

25.KuboM,NakamuraM,TasakiA,etal.:Hedgehogsignalingpathwayisa new therapeutic target for patients with breast cancer. Cancer Res 2004;64:6071-74.

26.Uemura,N.etal.:Helicobacterpyloriinfection and the developmentof gastriccancer.N EnglJMed2001;345:784-789.

27.HigashiH,TsutsumiR,Muto S,Sugiyama T,Azuma T,Asaka M, Hatakeyama M:SHP-2 tyrosine phosphatase as an intracellular targetof

HelicobacterpyloriCagA protein.Science2002;295:683-86.

28.BrandtS,KwokT,Hartig R,König W,BackertS:NF-κB activation and potentiation ofproinflammatory responsesby theHelicobacterpyloriCagA protein.ProNatlAcadSciU S A 2005;102:9300-05.

29.VermeulenL,DeWG,NotebaertS,VandenBW,HaegemanG:Regulation ofthetranscriptionalactivityofthenuclearfactor-κB p65subunit.Biochem. Pharmacol2002;64:963-70.

30.Chen F,Castranova V,ShiX:New insights into the role ofnuclear factor-κB incellgrowthregulation.Am JPathol2001;159:387-97.

31.Karin M,Cao Y,Greten FR,LiZW:NF-κB in cancer:from innocent bystandertomajorculprit.NatRevCancer2002;2:301-10.

32.KarinM,LinA:NF-κB atthecrossroadsoflifeanddeath.NatImmunol 2002;3:221-27.

(39)

33.SasakiN,MorisakiT,HashizumeK,Yao T,TsuneyoshiM,Noshiro H, Nakamura K,Yamanaka T,Uchiyama A,Tanaka M,Katano M:Nuclear factor-κB p65 (RelA) transcription factor is constitutively activated in humangastriccarcinomatissue.ClinCancerRes2001;7:4136-42.

34.Luo JL,Kamata H,Karin M:IKK/NF-κB signaling:balancing life and death.anew approachtocancertherapy.JClinInvest2005;115:2625-32. 35.JohnsonRL,ScottMP:Controlofcellgrowthandfateby patchedgenes.

ColdSpringHarborSympQuantBiol.1997;62:205-10.

36. Marigo V, Scott MP, Johnson RL, Goodrich LV, and Tabin CJ: Conservationinhedgehog signaling:inductionofachickenpatchedhomolog bySonichedgehoginthedevelopinglimb.1996;122:1225-30.

37.Goodrich LV,Jung D,Higgins KM,ScottMP:Overexpression ofPtc1 InhibitsInduction ofShh TargetGenesand PreventsNormalPatterning in theNeuralTube.199;211:323-34.

(40)

Abstract(InKorean)

위암상피세포에서 Helicobacterpylori감염에

따른 Sonichedgehog신호 조절

<지도교수 이 용 찬> 연세대학교 대학원 의과학과 최 연 정 최근 암의 발생과정은 분화가 끝난 세포에서 생기는 것이 아니라 각 장 기에 위치한 성체 줄기세포에서 생긴다는 보고들이 나오면서 정상적인 분 화에 관여되는 신호전달체계에 관심을 가지게 되었으며,여러 연구들을 통하여 정상적인 분화에 관여되는 신호전달과정이 발암과정에 관여됨이 보고 되었다.이중 대표적인 것이 Hedgehog (Hh)신호전달과정이다.만 성적인 자극이 발생,분화에 관여하는 신호체계를 비정상적으로 자극한다 는 개념에서 발암과정에 관여된다는 관점이나,실제로 이의 기전자체를 조사한 연구들은 거의 없다.따라서 이번 연구과제에서는 위의 대표적 만

(41)

성자극중의 하나인 Helicobacterpylori(H.pylori)감염이 위암발생에 있 어 Hh 신호전달과정에 미치는 영향을 밝힘으로써 성체 줄기세포에서의 암기원에 대한 기전 연구에 일익을 담당하고자 하였다.위암상피세포주 AGS,MKN45각각에 여러 종류의 H.pylori감염시켰다.H.pylori균주 로는 60190(cagPAIpositive),8822(cagPAInegative)그리고 60190균 주에 cagA,cagE 유전자를 각각 knock-out시킨 변이된 균주도 사용하 였다.H.pylori감염 후 위암상피세포주에서 Shh,Ptc1과 Gli1의 전사수 준에서의 조절과 단백질의 발현정도를 관찰하기 위해 RealtimeRT-PCR 과 westernblotting을 수행하였다.invivo연구로는 H.pylori감염이 확 인된 위암환자에서 추출한 위점막조직을 이용하여 면역화학염색 실시하였 고,H.pylori 감염여부에 따른 발현의 변화를 분석하였다.Western blotting과 RealtimeRT-PCR의 결과를 통해 H.pylori감염이 Shh의 발 현을 증가시키는 것을 알 수 있었다.게다가 cagA positive균주가 Shh의 발현을 더욱 증가시키는 것이 관찰되었다. Hh 신호전달과정에서 transcriptionaltarget인 Gli1의 단백질과 mRNA가 H.pylori감염시킨 후 증가하였다.이는 위암상피세포에서 H.pylori감염이 Hh신호전달과정을 활성화 시킨다는 것을 의미한다.in vivo 연구에서 위암환자로부터 얻은 위점막조직의 면역화학염색 결과,H.pylori감염 된 환자의 조직에서 H. pylori감염이 되지 않은 환자의 조직보다 Shh와 Gli1의 발현이 높았다. 이번 연구의 결과로 H.pylori감염과 Shh발현은 상호연관성이 있는 것 으로 밝혀졌다.H.pylori감염은 위암상피세포주에서 Shh발현을 증가시 킴으로서 Hh 신호전달과정을 활성화 시킨다.그리고 H.pylori의 중요 단백인 CagA가 위암상피세포주에서 Shh 발현에 영향을 미친다는 것을 알 수 있었다. 빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲빲 핵심되는 말 :위암,Hedgehog신호조절,Helicobacter.pylori