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Hindawi Publishing CorporationMediators of InflammationVolume 2013 Article ID 152860 11 pageshttpdxdoiorg1011552013152860

Research ArticleExpression of Annexin-A1 and Galectin-1 Anti-InflammatoryProteins and mRNA in Chronic Gastritis and Gastric Cancer

Yvana Cristina Jorge1 Mayra Mioto Mataruco1 Leandro Pires Arauacutejo1

Ana Flaacutevia Teixeira Rossi1 Juliana Garcia de Oliveira1 Marina Curado Valsechi1

Alaor Caetano2 Kenji Miyazaki3 Ceacutelia Sebastiana de Jesus Fazzio4

Jorge Alberto Thomeacute5 Paula Rahal1 Sonia Maria Oliani1 and Ana Elizabete Silva1

1 Department of Biology Sao Paulo State University (UNESP) Campus de Sao Jose do Rio Preto Rua Cristovao Colombo 226515054-000 Sao Jose do Rio Preto SP Brazil

2 Rio Preto Endoscopy Center Sao Jose do Rio Preto SP Brazil3 Endoscopy Service Hospital de Base Sao Jose do Rio Preto SP Brazil4 Legal Medicine Department and Pathology Service Hospital de Base Sao Jose do Rio Preto SP Brazil5 Institute of Anatomical Pathology and Cytopathology Sao Jose do Rio Preto SP Brazil

Correspondence should be addressed to Sonia Maria Oliani smolianiibilceunespbr andAna Elizabete Silva anabeteibilceunespbr

Received 7 May 2012 Accepted 28 December 2012

Academic Editor Eeva Moilanen

Copyright copy 2013 Yvana Cristina Jorge et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Objective The anti-inflammatory proteins annexin-A1 and galectin-1 have been associated with tumor progression This scenarioprompted us to investigate the relationship between the gene and protein expression of annexin-A1 (ANXA1AnxA1) and galectin-1(LGALS1Gal-1) in an inflammatory gastric lesion as chronic gastritis (CG) and gastric adenocarcinoma (GA) and its associationwithH pylori infectionMethods We analyzed 40 samples of CG 20 of GA and 10 of normal mucosa (C) by the quantitative real-time PCR (qPCR) technique and the immunohistochemistry assay Results HighANXA1mRNA expression levels were observed in90 (3640) of CG cases (mean relative quantification RQ= 426 plusmn 203) and in 80 (1620) of GA cases (mean RQ= 438 plusmn 477)However LGALS1mRNA levels were high (mean RQ= 244 plusmn 326) in 60 (1220) of the GA cases while low expression was foundin CG (mean RQ= 043 plusmn 313 119875 lt 001) Normal mucosa showed modest immunoreactivity in stroma but not in epitheliumwhile stroma and epithelium displayed an intense immunostaining in CG and GA for both proteins Conclusion These results haveprovided evidence that galectin-1 and mainly annexin-A1 are overexpressed in both gastritis and gastric cancer suggesting a strongassociation of these proteins with chronic gastric inflammation and carcinogenesis

1 Introduction

Chronic inflammation has been recognized as a process thatcan trigger cancer due to the host immune response withlocal expression of cytokines chemokines adhesion mole-cules and pro- and anti-inflammatory proteins that stimu-late processes such as proliferation survival cell migrationand neovascularization The strongest association betweenchronic inflammation and malignancy is observed in gastriccancer induced by Helicobacter pylori infection [1]

The inflammatory process resulting from H pylori infec-tion triggers a cascade of events initialized by chronicgastritis that evolves to gastric atrophy intestinal metaplasiadysplasia and finally carcinoma [2]This bacterium is presentin 90 of all chronic gastritis patients [3] and in 77 ofnoncardia gastric cancers [4]

Both the intrinsic factors of the host and the bacterialvirulence are associated with the development of gastric can-cer induced by H pylori Among the bacterial genes there iscagA which is detected in about 63 of patients with gastric

2 Mediators of Inflammation

cancer [5] The CagA protein is internalized by the host epi-thelial cells disrupting the cell cycle and inducing cell inva-sion through the activation of matrix metalloproteases [6]

Bacterial lipopolysaccharides activate several cell pro-cesses including expression of annexin-A1 (AnxA1) [7] andgalectin-1 (Gal-1) [8] which are both anti-inflammatory pro-teins During the inflammatory response AnxA1 is translo-cated from the cell cytoplasm to the membrane resultingin a decrease in the transmigration of inflammatory cellsto the site of injury [9] Furthermore AnxA1 plays its anti-inflammatory role by inhibiting the activities of phospholi-pase A2 and inducible nitric oxide synthase [10] It is alsoassociated with modifications of the cytoskeleton transportof molecules ion flux differentiation and migration cellgrowth and apoptosis [11]

Galectin-1 (Gal-1) a member of a family of carbohydrate-binding proteins may act in the same manner as AnxA1 inthe transmigration of inflammatory cells being importantalso in T-cell apoptosis by binding to T-cell receptors thustriggering the Fazcaspase cascade [12] It also contributesto different events associated with carcinogenesis includingtumor transformation cell cycle regulation apoptosis celladhesion migration and inflammation [13 14]

Nevertheless there are only a few studies in the liter-ature that evaluated the expression pattern of those anti-inflammatory mediators in gastric mucosa but their specificfunctions are unclear While in gastric cell lines the Gal-1protein expression increased [15 16] in gastric adenocarci-noma was observed low expression in tumor cells [17] Inturn AnxA1 was studied in gastric tissue with discrepantfindings such as increased expression during gastric mucosaldamage healing [18] loss of expression in metastatic gastriccancers [19] higher expression in diffuse-type gastric cancercompared to the intestinal type [20] and decreased expres-sion in gastric adenocarcinoma but with positive staining inadvanced stage and peritoneal dissemination [21]

Thus further research in this field might improve ourunderstanding of the possible role of those anti-inflammatoryproteins in carcinogenesis which is at present fairly limitedand controversial Moreover such studies may lead to thepossible identification of AnxA1 and Gal-1 as potentialbiomarkers in gastric cancer progression

Therefore the aim of this study was to investigate therelative expression levels of ANXA1 and LGALS1 mRNA byquantitative real-time PCR (qPCR) and the expression ofboth proteins by immunohistochemical assay in inflamma-tory gastric lesions such as chronic gastritis compared togastric cancer We also investigated a possible relationshipbetween mRNA expression levels andH pylori infection andits cagA virulence genotype in both lesions evaluated

2 Materials and Methods

21 Subjects and Samples Gastric biopsies were obtainedfrom seventy (70) individuals submitted to endoscopy at theEndoscopy Service of the Hospital de Base and at Rio PretoEndoscopy Center both in Sao Jose do Rio Preto SP BrazilThe histopathological data were supplied respectively by the

Legal Medicine Department and by the Pathology Serviceand the Institute ofAnatomical Pathology andCytopathology(IAPC) in the same city For each individual three (03)biopsy samples from the antrum region were collected formolecular studies and one for immunohistochemical analy-sis

The gastric adenocarcinoma (GA) group comprised 20individuals (14 male and 6 female mean age 634 plusmn 14 years)with a histopathologically confirmed diagnosis of gastric ade-nocarcinoma [22] Among the studied samples 12 cases werediagnosed as intestinal-type adenocarcinoma (IGC) and 8 asdiffuse-type adenocarcinoma (DGC) The chronic gastritis(CG) group was composed of 40 individuals (18 male and 22female mean age 525 plusmn 15 years) with a histopathologicallyconfirmed diagnosis of chronic gastritis [23]

The biopsies for the control (C) group were obtainedfrom 10 healthy individuals (7 male and 3 female meanage 35 plusmn 108 years) with no dyspeptic gastric complaintsand diagnosed as histopathologically normal gastric mucosaEpidemiological data on the study population were collectedusing a standard interviewer-administered questionnairewith questions about current and past occupation smokinghabits alcohol intake and family history of cancer None ofthe 70 subjects were under antibiotic or anti-inflammatorytreatment neither radiotherapy nor chemotherapy About60 of all patients in the GA group were smokers and 40were drinkers while all patients in the CG and C groupswere nonsmokers and nondrinkers Smokers were defined asindividuals who consumed at least 100 cigarettes during theirlifetime and alcohol consumers were those who drank morethan four times a week [24]

The Research Ethics Committee of the participating insti-tution approved this research (CEP IBILCEUNESP number05809) and written informed consent was obtained from allindividuals studied

22 Isolation of Total Nucleic Acids and Reverse-TranscriptionPCR (RT-PCR) Soon after collection the biopsies werestored in RNA later solution (Applied Biosystems) at minus20∘Cto preserve their integrity until RNA extraction The nucleicacid extraction was performed according to the protocolaccompanying the reagent TRIzol (Invitrogen) that allows thesimultaneous extraction of RNA and DNA RNA and DNAconcentrations were determined in a NanoDrop ND1000spectrophotometer (Uniscience) by measuring absorbance at260 and 280 nmDNA samples were stored atminus20∘C and usedfor the molecular diagnosis of H pylori

Afterwards reverse-transcription (RT) PCR was per-formed in an automated thermocycler using 25120583g of totalRNA in the presence of 125 120583L of oligo-d(T)16 (05120583g120583L)20 120583L of RNAse Inhibitor (80U120583L) and a High CapacitycDNA Archive Kit (Applied Biosystems) in a total volumeof 50120583L according to the manufacturerrsquos instructions Thereactions were carried out for 10 minutes at 25∘C followed by120 minutes at 37∘C The integrity of all cDNA preparationswas tested by a PCR assay of a 613 bp ACTB (120573-actin) genefragment used as control for abundant transcripts whoseprimer sequences were F 51015840-GGCATCGTGATGGACTCC-31015840 and R 31015840-GCTGGAAGGTGGACAGCG-51015840

Mediators of Inflammation 3

Table 1 Primers sequences used in multiplex PCR to determineHpylori infection and cagA genotype and in q-PCR assays

Gene Sequence 51015840ndash31015840

HpX F CTGGAGARACTAAGYCCTCCR GAGGAATACTCATTGCGAAGGCGA

CYP1A1 F CTCACCCCTGATGGTGCTATR TTTGGAAGTGCTCACAGCAG

cagA F ATGACTAACGAAACTATTGATCR CAGGATTTTTGATCGCTTTATT

ANXA1 F GCAGGCCTGGTTTATTGAAAR GCTGTGCATTGTTTCGCTTA

LGALS1 F GGACATCCTCCTGGACTCAR GTTGAAGCGAGGGTTGAAGT

ACTB F TGCCCTGAGGCACTCTTCR CGGATGTCCACGTCACAC

23 Molecular Diagnoses for H pylori-cagA To determinethe presence of H pylori infection DNA samples weresubjected to a multiplex PCR reaction containing primersfor the bacterial gene HpX [25] and for CYP1A1 (humanhousekeeping gene which attests the integrity of the DNA)

In summarywe used 50120583Lof 10Xbuffer 50120583Lof dNTPs(123mmolL Invitrogen) 20 120583LMgCl

2(25mmolL) 20 120583L

of primers (10 nmol120583L Invitrogen) 245120583L of dH2O 50120583L

of genomic DNA and 05 120583L of Taq DNA Polymerase(5U120583L Invitrogen) The material was processed in an auto-mated thermocycler and was initially subjected to a temper-ature of 94∘C for 5 minutes for denaturation Subsequentlyit was subjected to 40 amplification cycles at 94∘C for 45seconds at 60∘C for 30 seconds and at 72∘C for 90 secondsfollowed by a final extension cycle of 7 minutes at 72∘CThe amplification products were visualized on 20 agarosegel stained with ethidium bromide Fragments of 150 bp and226 bp corresponding to genesCYP1A1 andHpX respectivelywere observed

The H pylori-positive samples were then subjected to asecond PCR run to investigate the virulence genotype cagAof the bacterium [26] The parameters used were the same asfor the previous reaction except the annealing temperaturewhich in this case was 52∘C The product was visualized on10 agarose gel stained with ethidium bromide and a 232 bpfragment was observed Positive and negative controls wereused in all experiments The primer sequences are listed inTable 1

24 Quantitative Analysis of the Relative Amount of ANXA1and LGALS1 mRNA by Quantitative Real-Time PCR (q-PCR) The relative quantification q-PCR assay for ANXA1and LGALS1 mRNA expression was performed in an ABIPrism 7300 Sequence Detector System (Applied BiosystemsFoster City CA USA) according to the instructions for theSYBR Green PCR Core Reagent (Applied Biosystems) usingprimers specific for genes ANXA1 and LGALS1 [27] GeneACTBwas used as endogenous control (reference gene) of thereaction because it had shown the lowest variation compared

to 120572-tubulin and 1205732-microglobulin genes in a previous study[28] The primer sequences are presented in Table 1

The expression levels of the ANXA1 LGALS1 and ACTBmRNA were tested in triplicate (cDNA from the same RTreaction but in separated wells) Controls with no templatecDNA were used for each assay (negative control) Samplesof normal gastric mucosa were mixed to form a pool thatwas used as a calibrator (standard sample)The q-PCR assayswere performed in a total volume of 50 120583L containing 10 120583Lof SYBR Green Master Mix (Applied Biosystems) 25 ng ofcDNA and 04 120583M of ANXA1 and 05 120583M LGALS1 primers

After initial incubation at 50∘C for 2min to allow uracil-N-glycosylase (UNG) digestion and at 95∘C for 10min to acti-vate the AmpliTaq Gold DNA polymerase (both provided bythe Universal PCR Master Mix) the samples were amplifiedby subjecting them to 40 biphasic cycles of 95∘C for 15 sec and60∘C for 1min

The fluorescence signal was measured in the extensionphase of the PCR reaction and a threshold value (C

119879) of

fluorescence in the exponential part of the amplificationcurve was selected The larger the quantities of the materialat start the lower the CT values Relative quantification (RQ)of genes ANXA1 and LGALS1 was obtained as described byPfaffl [29] and normalized with the 120573-actin control referencegene and normal gastric mucosa The transcript levels wereconsidered to be upregulated if RQ gt 20

25 Immunohistochemistry Deparaffinized sections (4120583m)were incubated in citrate buffer pH 60 at 96∘C for 30minutes washed with distilled water incubated with 3hydrogen peroxide in methanol (30 minutes) and washedin phosphate-buffered saline (PBS pH 74) The primaryantibodies rabbit polyclonal anti-Gal-1 and rabbit polyclonalanti-ANXA1 (Zymed Laboratories Cambridge UK) werediluted to 1 500 or 1 2000 respectively in 1 bovine serumalbumin (BSA) and applied overnight at 4∘C As negativecontrols some sections were incubated with 1 BSA withoutany primary antibody Fragments were then washed in PBSincubated with the universal LSAB kitHRP secondary anti-body (Dako USA) according to the manufacturerrsquos protocolwashed in PBS and developedwith 331015840-diaminobenzidine inchromogen solution (Dako USA) The sections were washedthoroughly in distilled water counterstained with hema-toxylin and mounted on glass slides Densitometric analysisfor AnxA1 and Gal-1 immunostaining was performed usingan arbitrary scale from 0 to 255 with the AxioVision softwareon a Zeiss-Axioskop II light microscope and the data wereexpressed as mean plusmn SE

26 Statistical Analysis Fisherrsquos exact test was used to deter-mine if there were significant differences between groupsregarding the presence of bacteria and the cagA genotype thehistological type of tumor and the gender The data obtainedfrom mRNA quantification were expressed as meanplusmn SDTo assess differences in the mRNA relative expression levelsbetween the groups we used the nonparametric Mann-Whitney test To evaluate the association between geneexpression and the presence of the bacterium and cagA


Table 2 Distribution of infection byH pylori and cagA strains intochronic gastritis (CG) and gastric adenocarcinoma (GA) groups

Status CGN ()

GAN ()

H pyloriPositive 16 (40) 11 (55)Negative 24 (60) 9 (45)

Total 40 (100) 20 (100)119875 029Genotype cagA

Positive 10 (625) 3 (273)Negative 6 (375) 8 (727)

Total 16 (100) 11 (100)119875 012119873 number of individuals

genotype histological type of tumor gender smoking anddrinking we used the nonparametric 119905-test with Welchrsquoscorrection The value of protein expression was expressed asmeanplusmn SE The mean densitometry analysis results obtainedfor proteins AnxA1 and Gal-1 were compared by ANOVAfollowedmdashif significantmdashby the Bonferroni testThese analy-ses were performed using theGraphPad InStat andGraphPadPrism 4 software The value was considered significant if 119875 lt005

3 Results

31 Molecular Diagnoses for H pylori-cagA The frequenciesof cases with positive molecular diagnosis for H pylori andgenotype cagA in groups CG and GA are presented inTable 2 All 10 samples of normal mucosa were confirmed bymolecular testing as H pylori negative

Out of a total of 60 samples from the case groups 45were H pylori positive 40 (1640) in the CG group and55 (1120) in the GA group with no significant differencebetween the groups (119875 = 029) Regarding the genotype cagA48 of H pylori-positive samples were cagA positive 625(1016) in the CG and 273 (311) in the GA group Againthere was no significant difference between the groups (119875 =012)

32 Relative Gene Expression Analysis The relative expres-sion levels of ANXA1 mRNA after normalization with theACTB reference gene and comparison with the normalmucosa were increased in 90 (3640) of theCGcases (meanRQ = 426 plusmn 203) and in 80 (1620) of the GA cases (meanRQ = 438 plusmn 477) so there was no statistically significantdifference between the groups (119875 = 033) For LGALS1 themRNA relative expression values found were lower only theGA group showed overexpression in 60 (1220) of the cases(mean RQ= 244 plusmn 326) The CG group showed constitutiveexpression (mean RQ = 043 plusmn 313) with only 75 (340)of the cases presenting increased expression Thus the meanlevel of LGALS1 mRNA expression was significantly higher

in the GA than in the CG group (119875 lt 001) (Table 3 andFigure 1)

In another analysis we investigated the GA group fora possible association between ANXA1 and LGALS1 mRNAexpression and risk factors such drinking smoking and his-tological type of gastric cancer (Table 4) but no associationwas observed In addition comparing the variables genderH pylori infection and cagA+ genotype in the CG and theGA groups (Table 5) a significant difference was found inthe GA group for the gender and the mean level of ANXA1expression (119875 = 004) due to a 2 times greater expressionof this gene in the females than in the males of this groupLikewise the cagA+ genotype also showed an associationwith the ANXA1 expression level in the GA group due to ahigher mRNA expression in the cagA-positive compared tothe cagA-negative cases (meanRQ640 versus 277119875 lt 001)None of the other investigated factors appears to be associatedwith the levels of ANXA1 and LGALS1mRNA

33 Protein Expression Measured by ImmunohistochemistryAssay Protein expression was evaluated in normal mucosaCG intestinal (IGC) and diffuse- (DGC-) type gastric cancerModest expression of AnxA1 and Gal-1 was observed inthe stroma of normal mucosa while the epithelium did notshow any expression of these proteins (Figures 2(a) and 3(a)resp) However in the inflammatory process of CG mucosaintense immunostaining of AnxA1 and Gal-1 was seen inthe basal portion of the epithelium (Figures 2(b) and 3(b)resp) Positive immunostaining for AnxA1 and Gal-1 wasalso observed in DGC (Figures 2(c) and 3(c) resp) and IGC(Figures 2(d) and 3(d) resp) tumor cells In some areas ofgastric cancer samples it was possible to identify epithelialcells showing AnxA1 and Gal-1 expression

Themean optical densitometry values ofAnxA1 andGal-1expression are presented in Figures 2(e) and 3(e) respectivelyThe AnxA1 mean density was 9079 for normal mucosa (Cgroup) while for CG and GA it was respectively 16857 and19020 Thus a significant difference was found comparingthe normal mucosa with the CG and GA groups (119875 lt 001for both) The mean density of the Gal-1 protein was lower inall three groups (8652 13697 and 14630 in C CG and GAresp) showing a statistically significant difference betweenthe normal mucosa and the CG and GA groups (119875 lt 001 forboth)

In another densitometry analysis to determine the cyto-plasmic and nuclear immunoreactivity of AnxA1 separatelyin all groups although observed nuclear immunostainingmainly in gastric cancer the data obtained in our results didnot show a statistically significant difference between normalmucosa and CG (119875 = 019) and normal mucosa and GA(119875 = 018) (data not shown)

4 Discussion

In the present study we evaluated AnxA1 and Gal-1 anti-inflammatory proteins and mRNA expression in a group ofprecursor lesions such as chronic gastritis compared to gastriccancer and their association with risk factors Among the risk


Table 3 Comparison of ANXA1 and LGALS1mRNA relative expression levels between the chronic gastritis (CG) and the gastric adenocar-cinoma (GA) groups

Variable 1198601198731198831198601 1198711198661198601198711198781

CG GA CG GARelative expression (mean plusmn SD) 426 plusmn 203 438 plusmn 477 043 plusmn 313 244 plusmn 326

Minimum minus297 minus788 minus1078 minus695Maximum 890 929 1091 928119875 033 lt001lowastlowastSignificant difference

Table 4 Relative expression of ANXA1 and LGALS1 mRNA in thegastric adenocarcinoma group related to drinking smoking andhistological type of cancer

Variable 1198601198731198831198601 1198711198661198601198711198781

DrinkingYes 8 (40) 8 (40)(mean plusmn SD) 229 plusmn 576 174 plusmn 149

No 12 (60) 12 (60)(mean plusmn SD) 598 plusmn 261 282 plusmn 391

119875 015 039Smoking

Yes 12 (60) 12 (60)(mean plusmn SD) 401 plusmn 554 244 plusmn 218


119875 093 099Histology

Intestinal 12 (60) 12 (60)(mean plusmn SD) 582 plusmn 289 360 plusmn 279

Diffuse 8 (40) 8 (40)(mean plusmn SD) 155 plusmn 602 071 plusmn 332

119875 009 006

factors we investigated the presence of H pylori in bothlesions and its virulence genotype cagA since this bacteriumoften triggers the progression of the gastric carcinogenesiscascade To the best of our knowledge this is the first studythat has evaluated the expression of AnxA1 and Gal-1 inchronic gastritis

We have found a high relative expression of ANXA1mRNA already in the CG inflammatory process inwhich 90 of cases showed an increased expressionlevel (mean RQ = 426) which became 3 to 8 times higherafter normalization with the ACTB reference gene andcomparison with normal mucosa Similarly in the GA group80 of cases presented upregulated expression levels whichwere 3 to 9 times higher (mean RQ = 438) than that innormal mucosa For LGALS1 our study showed a slightlyincreased relative expression only in 60 of the GA cases(mean RQ = 244) with an increase of 2 to 7 times but inchronic gastritis the mean value was low (mean RQ = 043)In general the immunohistochemical analysis confirmedthe results of mRNA expression by qPCR although in CGthe relative expression levels of LGALS1 mRNA was notequivalently increased as protein expression

Studies on the expression of ANXA1mRNA in neoplasticprocesses are still limited and the results are conflictingFor example loss of expression was found in squamous cellcarcinoma of the esophagus [30] prostatic adenocarcinoma[31] sinonasal adenocarcinoma [32] larynx [33] and breastcancer [34] On the other hand overexpression has beenreported in colorectal adenocarcinoma [35] urothelial car-cinoma [36] lung adenocarcinoma [37] and oral cancers[38] thus suggesting that changes in the expression levels ofANXA1may be related to the tissue or tumor type

Martin et al [18] reported that normal gastric mucosashows weak expression of the protein AnxA1 while in theulcer healing process the expression was increased pro-moting the reduction of the ulcer In contrast Yu et al[19] observed overexpression of both gene and protein innormal mucosa but loss of expression in 64 of primarygastric tumors mainly correlated with advanced stage andmetastasisMore recently Zhu et al [21] observed that AnxA1protein is expressed in both gastric adenocarcinoma (45)and normal tissues (69) but with different subcellulardistribution Similar results were reported byCheng et al [39]that observed high AnxA expression both mRNA and pro-tein associated with metastasis invasion and poor survivalin gastric cancer patients The authors also proposed a newmechanism of how AnxA1 regulates the gastric cancer cellinvasion through activation FPRERKITGB1BP1 pathway

In the present study the immunohistochemical analysisfor AnxA1 showed modest immunostaining in stromal cellsof normal mucosa and intense expression in stroma andepithelium of both CG and GA thus confirming the resultsof the mRNA expression analysis The assay used in thisstudy did not allow a clear differentiation of the cellularlocalization of the protein although positive immunostainingwas observed in the cytoplasm of epithelial cells and thenucleus of cancer cells with lower intensity and frequency inchronic gastritis However when the densitometry analysisto determine cytoplasmic and nuclear immunoreactivity ofAnxA1 separately was performed the data obtained didnot show a statistically significant difference among nor-mal mucosa chronic gastritis and gastric adenocarcinomaAlthough there are some reports that show translocationof AnxA1 during carcinogenesis as Alves et al [40] thatshowed 875 positivity for AnxA1 in larynx tumors andincreased immunoreactivity in the membrane compared tothe cytoplasm and the nucleus However compared to thenormal tissue the nuclear and cytoplasmic expression waslower In esophageal carcinoma Liu et al [41] found AnxA1


Table 5 Relative expression of ANXA1 and LGALS1mRNA in chronic gastritis (CG) and gastric adenocarcinoma (GA) groups according togender infection by H pylori and presence of cagA+ genotype

Variable 1198601198731198831198601 1198711198661198601198711198781

CG GA CG GAGender

Female 22 (55) 6 (30) 22 (55) 6 (30)(mean plusmn SD) 440 plusmn 180 667 plusmn 200 029 plusmn 355 481 plusmn 326

Male 28 (45) 14 (70) 28 (45) 14 (70)(mean plusmn SD) 408 plusmn 232 325 plusmn 516 060 plusmn 261 166 plusmn 296

119875 063 004lowast 075 010H pylori

Positive 16 (40) 11 (55) 16 (40) 11 (55)(mean plusmn SD) 404 plusmn 259 391 plusmn 548 minus005 plusmn 397 333 plusmn 316

Negative 24 (60) 9 (45) 24 (60) 9 (45)(mean plusmn SD) 440 plusmn 160 435 plusmn 405 075 plusmn 246 136 plusmn 323

119875 047 061 024 073Genotype cagA



119875 014 lt001lowast 009 051lowastSignificant difference

translocation from the cellular to the nuclear membraneWhile in gastric adenocarcinoma AnxA1 showed positivenuclear staining correlated with advanced disease stage andperitoneal dissemination but in normal tissues was predom-inantly localized in the cytoplasm [21] In addition weaknuclear staining also occasionally occurred in sporadic cellsof gastric tumors (14118 cases) evaluated by Cheng et al[39] But in general the AnxA1 immunostaining was mainlycytoplasmatic in the epithelial cells

Changes in the expression pattern of AnxA1 must berelated with factors that influence its translocation andexport such as the cellular concentration of calcium consid-ering that when the intracellular calcium level is increasedAnxA1 is translocated to membranes [42] Furthermore it isreported that protein phosphorylation is also required for thisprocess [20] In tumor cells calcium and other factorsmay bealtered resulting in an abnormal location ofAnxA1Howeverthis relationship needs to be better understood and may bethe connection between inflammation signal transductiondifferentiation and cellular transport in cancer [40]

To date Gal-1 is involved in various important aspects ofcarcinogenesis so mRNA and protein expressions have beenexamined in several types of cancer Overexpression has beenreported in colorectal cancer [43] Hodgkinrsquos lymphoma [44]squamous cell carcinomas of the larynx and carcinomas ofthe hypopharynx [45] Conversely there is also a report of lowexpression as in chronic inflammation of nasal polyposis[27]

Two research groups evaluated the expression of Gal-1 in gastric cell lines and both agreed that this protein isoverexpressed in tumor cells Chen et al [16] showed higherexpression of the protein in TMC-1 compared to SC-M1 cellsproposing Gal-1 as a biomarker for metastasis Lim et al [15]

investigatedAGS cells infected byH pylori and observed highexpression levels of Gal-1 in this cell type Recently Estofoleteet al [46] observed by immunostaining that both Gal-1and -3 were highly expressed in an experimental model ofN-methyl-N1015840-nitro-N-nitrosoguanidine- (MNNG-) inducedgastric carcinogenesis

The immunohistochemistry assay performed showedmodest staining for Gal-1 in the stroma of normal mucosawhile in CG and GA the staining was stronger in stromaand epithelium In contrary the LGALS1 mRNA levels werelower in the CG group in comparison with the GA groupSeveral studies have shown lack of correlation between RNAexpression and protein expression profiles using differentmethodologies Some authors state that in fact the use ofmRNA expression patterns is insufficient for understandingthe expression of protein products as additional posttran-scriptional mechanisms including protein translation post-translational modification and degradation may influencethe level of a protein present in a given cell or tissue [47ndash50]

The comparison between the CG and GA groups regard-ing ANXA1 and LGALS1 mRNA levels and risk factors didnot show any association with the LGALS1 mRNA levelsHowever a positive association was observed between over-expression of ANXA1 and female gender in the GA groupsince in women the mRNA expression was twice as high as inmen (mean RQ= 667 versus 325) and the H pylori-cagA+infection showed an mRNA mean level approximately twicehigher in patients infected by cagA+ strains (mean RQ= 640versus mean RQ = 277) Despite the relevance of the studyit should be considered some limitations due to the reducednumber of cases in the subgroup stratification which shouldbe interpreted with cautions It would need confirmation infurther studies with larger population


Chronic gastritis (CG)

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(a)

Gastric adenocarcinoma (GA)

Valu

es o

f re

lativ

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pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(b)


LGALS1

Valu

es o

f re

lativ

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sion

(log2

)

5

10

15

0

minus5

minus15

minus10

(c)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

1210

86420

minus2minus4minus6minus8

(d)

Mea

ns o

f va

lues

of

rela

tive

expr

essio

n (lo

g2)

10

75

5

25

0CG GA

ANXA1LGALS1

(e)

Figure 1 Values of relative gene expression (log2) of ANXA1 (a b) and LGALS1 (c d) and means of values of relative gene expression (e) inthe chronic gastritis (CG) and gastric adenocarcinoma (GA) groups

It is well known that there is sexual dimorphism in theimmune and inflammatory responses in humans Womenproduce more vigorous cellular and humoral reactions aremore resistant to certain infections and suffer a higherincidence of autoimmune diseases than males [51] It ispossible that hormonal differences may explain part of thisdimorphism It has also been suggested that the ANXA1expression may differ in several types of cancer due tohormonal influence [52]

Ang et al [52] conducted a very elegant study on the influ-ence of AnxA1 in MCF-7 breast cancer cells in which theyshowed that 17 120573-estradiol (active metabolite of estrogen)regulates ANXA1 expression Moreover 17 120573-estradiol was

shown to activate cyclic-AMP- (cAMP-) responsive element(CRE) binding (CREB) proteins to induce a transcriptionalactivity The promoter region of ANXA1 was examined andfound to contain a similar near identical 8-nucleotidesequence (TGATGTCA) to the CRE consensus sequence(TGACGTCA) So estrogen could also promote the tran-scription of ANXA1 Yet those authors proposed anothertheory to explain the connection between estrogen andAnxA1 levels Elevated 17 120573-estradiol activates the ERK12pathway increasing cell proliferation and this serves as asign to stimulate ANXA1 transcription in order to reducethe proliferative state Then AnxA1 upregulates p21 whichhas antiproliferative properties and reduces the proliferation


(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200

250

150

100

50

0CGC GA

AnxA1

lowast

lowastlowast

(e)

Figure 2 Endogenous annexin-A1 (ANXA1) expression in the gastric mucosa Immunoreactivity of ANXA1 in sections of gastric mucosatissue by rabbit polyclonal antibody ANXA1 (a) Histological analysis showingmodest immunoreactivity in stroma and negative for epithelialcells in normal mucosa Note the immunopositivity in basal portion of epithelial in (b) chronic gastritis and intense stromal-epithelialimmunostaining in (c) diffuse-type adenocarcinoma and (d) intestinal-type adenocarcinoma Hematoxylin counterstain Bar 20 120583m (e)Densitometry analyses on the gastric mucosa tissues immunostained for AnxA1 in normal mucosa (C) chronic gastritis (CG) and gastricadenocarcinoma (GA) groups Comparison between the C group and the CG (lowast) and GA (lowastlowast) groups was made by the ANOVA test with119875 lt 001

caused by the activation of ERK12 by estrogen Howeverin our study in the GA group the mean age of womenwas elevated (674 plusmn 184 years) which characterizes thepostmenopausal phase that have decreased estrogen levelsthus not justifying the relation between increased expressionofANXA1 and estrogenTherefore further studies are neededin order to clarify this issue

The relationship between the presence of virulence factorCagA and gastric carcinogenesis is well documentedWestern

populations infected with CagA-positive strains generallyhave an accentuated inflammatory response with increasedrisk of developing peptic ulcer and stomach cancer [53]The phosphorylation of the CagA protein activates SHP-2 (protein tyrosine phosphatase) which then inhibits theFAK (focal adhesion kinase) an enzyme that modulatesadhesion migration and cell survival [4] Consequentlythe decline in the activity of FAK triggers a rearrangementof the cytoskeleton known as the hummingbird phenotype


(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200 Gal-1

100

0CGC GA

lowast

lowastlowast

(e)

Figure 3 Endogenous galectin 1 (Gal-1) expression in the gastric mucosa Immunoreactivity of Gal-1 in sections of gastric mucosatissue by rabbit polyclonal anti-Gal-1 (a) Negative immunostaining in epithelium and modest positivity in stroma in normal mucosaImmunopositivity in basal portion of epithelial in chronic gastritis (b) and intense stromal-epithelial immunostaining in diffuse-typeadenocarcinoma (c) and intestinal-type adenocarcinoma (d) Hematoxylin counterstain Bar 20 120583m (e) Densitometry analyses on thegastric mucosa tissues immunostained for Gal-1 in normal mucosa (C) chronic gastritis (CG) and gastric adenocarcinoma (GA) groupsComparison between the C group and the CG (lowast) and GA (lowastlowast) groups was made by the ANOVA test with 119875 lt 001

characterized by elongation and spreading of host cells [54]Furthermore CagA participates in cell signaling by activatingthe PIK3CA and KRAS pathways [1] ERK (MAPK) [55 56]and MEKERK and JAK1 signaling pathway in gastric cancercells [57] To our knowledge there are no reports about therelationship to the CagA virulence factor and the expressionof ANXA1 and Gal-1 anti-inflammatory proteins HoweverLin et al [58] observed overexpression of AnxA4 in tumorcells of patients infected with H pylori and in gastric cancerSCM-1 cells after H pylori infection Recently Lin et al [59]

observed that infection by H pylori induced a change inAnxA1 and AnxA4 localization causing a translocation fromthe cytoplasm to the plasma membrane probably for epithe-lial cell membrane repair in the consequence of H pylori-generated membrane disruptions So due to the action ofCagA bacterial protein in different cell signaling pathways itis possible that it may also contribute to activation of ANXA1expression mainly considering that this protein plays a keyrole as intracellular Ca2+ flux modifications of the cytoskele-ton differentiation andmigration cell growth and apoptosis


5 Conclusions

In conclusion this study showed overexpression of bothANXA1mRNA and protein already in a precursor lesion suchas CG similar to GA in which higher expression levels wereobserved in H pylori-cagA+ cases suggesting upregulationof this gene in early stages of gastric carcinogenesis Inturn LGALS1 (mRNA or protein) was slightly overexpressedin both lesions indicating also its participation in gastriccarcinogenesis However as in several types of cancers therole of these proteins is not yet fully understood furtherinvestigations are needed to help clarify the molecular mech-anisms by which they act in this kind of lesion

Acknowledgment

This study was supported by the Brazilian agencies FAPESPCNPq and CAPES

References

[1] T Chiba HMarusawa and TUshijima ldquoInflammation-associ-ated cancer development in digestive organs mechanisms androles for genetic and epigenetic modulationrdquo Gastroenterologyvol 143 pp 550ndash563 2012

[2] P Correa ldquoA human model of gastric carcinogenesisrdquo CancerResearch vol 48 no 13 pp 3554ndash3560 1988

[3] T Robbins and R S Cotran ldquoStomachrdquo in Pathologic Bases ofDiseases pp 787ndash801 Saunders Philadelphia Pa USA 6th edi-tion 2005

[4] L T Nguyen T Uchida K Murakami T Fujioka and MMoriyama ldquoHelicobacter pylori virulence and the diversity ofgastric cancer in Asiardquo Journal of Medical Microbiology vol 57no 12 pp 1445ndash1453 2008

[5] J Q Huang G F Zheng K Sumanac E J Irvine and R HHunt ldquoMeta-analysis of the relationship between cagA seropos-itivity and gastric cancerrdquo Gastroenterology vol 125 no 6 pp1636ndash1644 2003

[6] H Isomoto Y Nishi K Ohnita et al ldquoThe relationship betweenplasma and gastric ghrelin levels and strain diversity in Heli-cobacter pylori virulencerdquo American Journal of Gastroenterol-ogy vol 100 no 6 pp 1425ndash1427 2005

[7] C D John F N Gavins N A Buss P O Cover and J CBuckingham ldquoAnnexinA1 and the formyl peptide receptor fam-ily neuroendocrine and metabolic aspectsrdquo Current Opinion inPharmacology vol 8 no 6 pp 765ndash776 2008

[8] J Almkvist and A Karlsson ldquoGalectins as inflammatory medi-atorsrdquoGlycoconjugate Journal vol 19 no 7-9 pp 575ndash581 2002

[9] T S Gastardelo A S Damazo J Dalli R J Flower M Perrettiand S M Oliani ldquoFunctional and ultrastructural analysis ofannexin A1 and its receptor in extravasating neutrophils duringacute inflammationrdquoAmerican Journal of Pathology vol 174 no1 pp 177ndash183 2009

[10] L Parente and E Solito ldquoAnnexin 1 more than an anti-phos-pholipase proteinrdquo Inflammation Research vol 53 no 4 pp125ndash132 2004

[11] V Gerke C E Creutz and S E Moss ldquoAnnexins linking Ca2+signalling to membrane dynamicsrdquo Nature Reviews MolecularCell Biology vol 6 no 6 pp 449ndash461 2005

[12] PMatarrese A Tinari EMormone et al ldquoGalectin-1 sensitizesresting human T lymphocytes to Fas (CD95)-mediated cell

death via mitochondrial hyperpolarization budding and fis-sionrdquo Journal of Biological Chemistry vol 280 no 8 pp 6969ndash6985 2005

[13] G A Rabinovich ldquoGalectin-1 as a potential cancer targetrdquoBritish Journal of Cancer vol 92 no 7 pp 1188ndash1192 2005

[14] C D Gil D Cooper G Rosignoli M Perretti and S M OlianildquoInflammation-inducedmodulation of cellular galectin-1 and -3expression in amodel of rat peritonitisrdquo Inflammation Researchvol 55 no 3 pp 99ndash107 2006

[15] J W Lim H Kim and K H Kim ldquoCell adhesion-related geneexpression by Helicobacter pylori in gastric epithelial AGScellsrdquo International Journal of Biochemistry and Cell Biology vol35 no 8 pp 1284ndash1296 2003

[16] Y R Chen H F Juan H C Huang et al ldquoQuantitative pro-teomic and genomic profiling reveals metastasis-related proteinexpression patterns in gastric cancer cellsrdquo Journal of ProteomeResearch vol 5 no 10 pp 2727ndash2742 2006

[17] S Bektas B Bahadir BHUcan and SOOzdamar ldquoCD24 andgalectin-1 expressions in gastric adenocarcinoma and clinico-pathologic significancerdquo Pathology and Oncology Research vol16 no 4 pp 569ndash577 2010

[18] G R Martin M Perretti R J Flower and J L Wallace ldquoAn-nexin-1 modulates repair of gastric mucosal injuryrdquo AmericanJournal of Physiology vol 294 no 3 pp G764ndashG769 2008

[19] G Yu J Wang Y Chen et al ldquoTissue microarray analysis re-veals strong clinical evidence for a close association betweenloss of annexin A1 expression and nodal metastasis in gastriccancerrdquo Clinical and Experimental Metastasis vol 25 no 7 pp695ndash702 2008

[20] C M Wu Y S Lee T H Wang et al ldquoIdentification of dif-ferential gene expression between intestinal and diffuse gastriccancer using cDNA microarrayrdquo Oncology Reports vol 15 no1 pp 57ndash64 2006

[21] F Zhu C Xu Z Jiang et al ldquoNuclear localization of annexin A1correlates with advanced disease and peritoneal disseminationin patients with gastric carcinomardquoAnatomical Record vol 293no 8 pp 1310ndash1314 2010

[22] P Lauren ldquoThe two histological main types of gastric carci-noma diffuse and so-called intestinal-type carcinomardquo ActaPathologica et Microbiologica Scandinavica vol 64 pp 31ndash491965

[23] M F Dixon R M Genta J H Yardley et al ldquoClassificationand grading of Gastritis the updated Sydney systemrdquoAmericanJournal of Surgical Pathology vol 20 no 10 pp 1161ndash1181 1996

[24] S AAhrendt J T Chow S C Yang et al ldquoAlcohol consumptionand cigarette smoking increase the frequency of p53 mutationsin non-small cell lung cancerrdquo Cancer Research vol 60 no 12pp 3155ndash3159 2000

[25] L L Gatti R de Labio L C da SilvaMDA C Smith and S LM Payao ldquocagA positive Helicobacter pylori in Brazilian chil-dren related to chronic gastritisrdquo Brazilian Journal of InfectiousDiseases vol 10 no 4 pp 254ndash258 2006

[26] R M Peek and M J Blaser ldquoHelicobacter pylori and gastroin-testinal tract adenocarcinomasrdquo Nature Reviews Cancer vol 2no 1 pp 28ndash37 2002

[27] A A S Sena P J S Provazzi A M Fernandes P M Cury PRahal and S M Oliani ldquoSpatial expression of two anti-inflam-matory mediators annexin 1 and galectin-1 in nasal polyposisrdquoClinical and Experimental Allergy vol 36 no 10 pp 1260ndash12672006

[28] M C Duarte E Babeto K R M Leite et al ldquoExpression ofTERT in precancerous gastric lesions compared to gastric can-cerrdquo Brazilian Journal of Medical and Biological Research vol44 no 2 pp 100ndash104 2011


[29] M W Pfaffl ldquoA new mathematical model for relative quantifi-cation in real-time RT-PCRrdquoNucleic Acids Research vol 29 no9 article e45 2001

[30] M Moghanibashi F R Jazii Z S Soheili et al ldquoProteomicsof a new esophageal cancer cell line established from Persianpatientrdquo Gene vol 500 pp 124ndash133 2012

[31] J S Kang B F Calvo S J Maygarden L S Caskey J LMohler andDKOrnstein ldquoDysregulation of annexin I proteinexpression in high-grade prostatic intraepithelial neoplasia andprostate cancerrdquo Clinical Cancer Research vol 8 no 1 pp 117ndash123 2002

[32] J P Rodrigo J M Garcıa-Pedrero J L Llorente et al ldquoDown-regulation of annexin A1 and A2 protein expression in intes-tinal-type sinonasal adenocarcinomasrdquo Human Pathology vol42 no 1 pp 88ndash94 2011

[33] R Silistino-Souza F C Rodrigues-Lisoni PM Cury et al ldquoAn-nexin 1 differential expression in tumor and mast cells inhuman larynx cancerrdquo International Journal of Cancer vol 120no 12 pp 2582ndash2589 2007

[34] C K Yom W Han S-W Kim et al ldquoClinical significance ofsnnexin A1 expression in breast cancerrdquo Journal Breast of Can-cer vol 14 pp 262ndash268 2011

[35] N Su X-Y Xu H Chen et al ldquoIncreased expression of annexinA1 is correlated with K-ras mutation in colorectal cancerrdquoTohoku Journal of Experimental Medicine vol 222 no 4 pp243ndash250 2010

[36] W-Y Kang W-T Chen Y-C Huang Y-C Su and C-Y ChaildquoOverexpression of annexin 1 in the development and differen-tiation of urothelial carcinomardquo Kaohsiung Journal of MedicalSciences vol 28 pp 145ndash150 2012

[37] Y F Liu P F Zhang M Y Li Q Q Li and Z C Chen ldquoIdenti-fication of annexin A1 as a proinvasive and prognostic factor forlung adenocarcinomardquo Clinical and Experimental Metastasisvol 28 no 5 pp 413ndash425 2011

[38] C Y Lin Y M Jeng H Y Chou et al ldquoNuclear localization ofannexin A1 is a prognostic factor in oral squamous cell carci-nomardquo Journal of Surgical Oncology vol 97 no 6 pp 544ndash5502008

[39] T-Y Cheng M-S Wu J-T Lin et al ldquoAnnexin A1 is associatedwith gastric cancer survival and promotes gastric cancer cellinvasiveness through the Formyl Peptide ReceptorExtracellu-lar Signal-Regulated KinaseIntegrin Beta-1-Binding Protein 1Pathwayrdquo Cancer vol 118 no 23 pp 5757ndash5767 2012

[40] VAAlves S Nonogaki PMWunsch-Filho et al ldquoAnnexinA1subcellular expression in laryngeal squamous cell carcinomardquoHistopathology vol 53 pp 715ndash727 2008

[41] Y LiuH XWangN Lu et al ldquoTranslocation of annexin I fromcellular membrane to the nuclear membrane in human eso-phageal squamous cell carcinomardquo World Journal of Gastroen-terology vol 9 no 4 pp 645ndash649 2003

[42] K Monastyrskaya E B Babiychuk A Hostettler U RescherandADraeger ldquoAnnexins as intracellular calcium sensorsrdquoCellCalcium vol 41 no 3 pp 207ndash219 2007

[43] T H Sheng L X Rong Z Y Li J Bo and S Lei ldquoTissue andserum galectin-1 expression in patients with colorectal carci-nomardquo Hepatogastroenterology vol 59 pp 389ndash394 2012

[44] P Kamper M Ludvigsen K Bendix et al ldquoProteomic analysisidentifies galectin-1 as a predictive biomarker for relapsedre-fractory disease in classical Hodgkin lymphomardquo Blood vol 117no 24 pp 6638ndash6649 2011

[45] S Saussez C Decaestecker F Lorfevre et al ldquoIncreased expres-sion and altered intracellular distribution of adhesiongrowth-regulatory lectins galectins-1 and -7 during tumour progression

in hypopharyngeal and laryngeal squamous cell carcinomasrdquoHistopathology vol 52 no 4 pp 483ndash493 2008

[46] C F Estofolete S Zucoloto S M Oliani A C Polli-Lopes andC D Gil ldquoMyenteric denervation downregulates galectin-1 and-3 expression in gastric carcinogenesisrdquo Digestive Diseases andSciences vol 56 no 6 pp 1637ndash1644 2011

[47] G Chen T G Gharib C C Huang et al ldquoDiscordant proteinand mRNA expression in lung adenocarcinomasrdquoMolecular ampCellular Proteomics vol 1 no 4 pp 304ndash313 2002

[48] D Greenbaum C Colangelo K Williams and M GersteinldquoComparing protein abundance and mRNA expression levelson a genomic scalerdquo Genome Biology vol 4 no 9 article 1172003

[49] Y Guo P Xiao S Lei et al ldquoHow is mRNA expression pre-dictive for protein expression A correlation study on humancirculating monocytesrdquo Acta Biochimica et Biophysica Sinicavol 40 no 5 pp 426ndash436 2008

[50] F E Rosa S M Silveira C G T Silveira et al ldquoQuantitativereal-time RT-PCR and chromogenic in situ hybridization pre-cise methods to detect HER-2 status in breast carcinomardquo BMCCancer vol 9 article 90 2009

[51] A Bouman M Jan Heineman andM M Faas ldquoSex hormonesand the immune response in humansrdquo Human ReproductionUpdate vol 11 no 4 pp 411ndash423 2005

[52] E Z F Ang H T Nguyen H L Sim T C Putti and L H KLim ldquoAnnexin-1 regulates growth arrest induced by high levelsof estrogen in MCF-7 breast cancer cellsrdquo Molecular CancerResearch vol 7 no 2 pp 266ndash274 2009

[53] J GKusters AHMVanVliet andE J Kuipers ldquoPathogenesisof Helicobacter pylori infectionrdquo Clinical Microbiology Reviewsvol 19 no 3 pp 449ndash490 2006

[54] R Tsutsumi H Higashi M Higuchi M Okada and MHatakeyama ldquoAttenuation of Helicobacter pylori CagAsdotSHP-2 signaling by interaction between CagA and C-terminal Srckinaserdquo Journal of Biological Chemistry vol 278 no 6 pp3664ndash3670 2003

[55] C C Allison T A Kufer E Kremmer M Kaparakis and R LFerrero ldquoHelicobacter pylori induces MAPK phosphorylationand AP-1 activation via a NOD1-dependent mechanismrdquo Jour-nal of Immunology vol 183 no 12 pp 8099ndash8109 2009

[56] J J Yang L Y Cho S H Ma et al ldquoOncogenic CagA promotesgastric cancer risk via activating ERK signaling pathways anested case-control studyrdquo PLoS ONE vol 6 no 6 Article IDe21155 2011

[57] J Zhou Y Xie Y Zhao S Wang and Y Li ldquoHuman gastrinmRNA expression up-regulated by Helicobacter pylori CagAthrough MEKERK and JAK2-signaling pathways in gastriccancer cellsrdquo Gastric Cancer vol 14 pp 322ndash331 2011

[58] L L Lin C N ChenW C Lin et al ldquoAnnexin A4 a novel mo-lecular marker for gastric cancer with Helicobacter pyloriinfection using proteomics approachrdquo Proteomics vol 2 no 4pp 619ndash634 2008

[59] L L Lin H C Huang S Ogihara et al ldquoHelicobacter pyloridisrupts host cell membranes initiating a repair response andcell proliferationrdquo International Journal of Molecular Sciencesvol 13 pp 10176ndash11092 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


cancer [5] The CagA protein is internalized by the host epi-thelial cells disrupting the cell cycle and inducing cell inva-sion through the activation of matrix metalloproteases [6]

Bacterial lipopolysaccharides activate several cell pro-cesses including expression of annexin-A1 (AnxA1) [7] andgalectin-1 (Gal-1) [8] which are both anti-inflammatory pro-teins During the inflammatory response AnxA1 is translo-cated from the cell cytoplasm to the membrane resultingin a decrease in the transmigration of inflammatory cellsto the site of injury [9] Furthermore AnxA1 plays its anti-inflammatory role by inhibiting the activities of phospholi-pase A2 and inducible nitric oxide synthase [10] It is alsoassociated with modifications of the cytoskeleton transportof molecules ion flux differentiation and migration cellgrowth and apoptosis [11]

Galectin-1 (Gal-1) a member of a family of carbohydrate-binding proteins may act in the same manner as AnxA1 inthe transmigration of inflammatory cells being importantalso in T-cell apoptosis by binding to T-cell receptors thustriggering the Fazcaspase cascade [12] It also contributesto different events associated with carcinogenesis includingtumor transformation cell cycle regulation apoptosis celladhesion migration and inflammation [13 14]

Nevertheless there are only a few studies in the liter-ature that evaluated the expression pattern of those anti-inflammatory mediators in gastric mucosa but their specificfunctions are unclear While in gastric cell lines the Gal-1protein expression increased [15 16] in gastric adenocarci-noma was observed low expression in tumor cells [17] Inturn AnxA1 was studied in gastric tissue with discrepantfindings such as increased expression during gastric mucosaldamage healing [18] loss of expression in metastatic gastriccancers [19] higher expression in diffuse-type gastric cancercompared to the intestinal type [20] and decreased expres-sion in gastric adenocarcinoma but with positive staining inadvanced stage and peritoneal dissemination [21]

Thus further research in this field might improve ourunderstanding of the possible role of those anti-inflammatoryproteins in carcinogenesis which is at present fairly limitedand controversial Moreover such studies may lead to thepossible identification of AnxA1 and Gal-1 as potentialbiomarkers in gastric cancer progression

Therefore the aim of this study was to investigate therelative expression levels of ANXA1 and LGALS1 mRNA byquantitative real-time PCR (qPCR) and the expression ofboth proteins by immunohistochemical assay in inflamma-tory gastric lesions such as chronic gastritis compared togastric cancer We also investigated a possible relationshipbetween mRNA expression levels andH pylori infection andits cagA virulence genotype in both lesions evaluated

2 Materials and Methods

21 Subjects and Samples Gastric biopsies were obtainedfrom seventy (70) individuals submitted to endoscopy at theEndoscopy Service of the Hospital de Base and at Rio PretoEndoscopy Center both in Sao Jose do Rio Preto SP BrazilThe histopathological data were supplied respectively by the

Legal Medicine Department and by the Pathology Serviceand the Institute ofAnatomical Pathology andCytopathology(IAPC) in the same city For each individual three (03)biopsy samples from the antrum region were collected formolecular studies and one for immunohistochemical analy-sis

The gastric adenocarcinoma (GA) group comprised 20individuals (14 male and 6 female mean age 634 plusmn 14 years)with a histopathologically confirmed diagnosis of gastric ade-nocarcinoma [22] Among the studied samples 12 cases werediagnosed as intestinal-type adenocarcinoma (IGC) and 8 asdiffuse-type adenocarcinoma (DGC) The chronic gastritis(CG) group was composed of 40 individuals (18 male and 22female mean age 525 plusmn 15 years) with a histopathologicallyconfirmed diagnosis of chronic gastritis [23]

The biopsies for the control (C) group were obtainedfrom 10 healthy individuals (7 male and 3 female meanage 35 plusmn 108 years) with no dyspeptic gastric complaintsand diagnosed as histopathologically normal gastric mucosaEpidemiological data on the study population were collectedusing a standard interviewer-administered questionnairewith questions about current and past occupation smokinghabits alcohol intake and family history of cancer None ofthe 70 subjects were under antibiotic or anti-inflammatorytreatment neither radiotherapy nor chemotherapy About60 of all patients in the GA group were smokers and 40were drinkers while all patients in the CG and C groupswere nonsmokers and nondrinkers Smokers were defined asindividuals who consumed at least 100 cigarettes during theirlifetime and alcohol consumers were those who drank morethan four times a week [24]

The Research Ethics Committee of the participating insti-tution approved this research (CEP IBILCEUNESP number05809) and written informed consent was obtained from allindividuals studied

22 Isolation of Total Nucleic Acids and Reverse-TranscriptionPCR (RT-PCR) Soon after collection the biopsies werestored in RNA later solution (Applied Biosystems) at minus20∘Cto preserve their integrity until RNA extraction The nucleicacid extraction was performed according to the protocolaccompanying the reagent TRIzol (Invitrogen) that allows thesimultaneous extraction of RNA and DNA RNA and DNAconcentrations were determined in a NanoDrop ND1000spectrophotometer (Uniscience) by measuring absorbance at260 and 280 nmDNA samples were stored atminus20∘C and usedfor the molecular diagnosis of H pylori

Afterwards reverse-transcription (RT) PCR was per-formed in an automated thermocycler using 25120583g of totalRNA in the presence of 125 120583L of oligo-d(T)16 (05120583g120583L)20 120583L of RNAse Inhibitor (80U120583L) and a High CapacitycDNA Archive Kit (Applied Biosystems) in a total volumeof 50120583L according to the manufacturerrsquos instructions Thereactions were carried out for 10 minutes at 25∘C followed by120 minutes at 37∘C The integrity of all cDNA preparationswas tested by a PCR assay of a 613 bp ACTB (120573-actin) genefragment used as control for abundant transcripts whoseprimer sequences were F 51015840-GGCATCGTGATGGACTCC-31015840 and R 31015840-GCTGGAAGGTGGACAGCG-51015840












2(25mmolL) 20 120583L









119879) of






Status CGN ()

GAN ()






3 Results









4 Discussion




Variable 1198601198731198831198601 1198711198661198601198711198781




Variable 1198601198731198831198601 1198711198661198601198711198781



119875 015 039Smoking



119875 093 099Histology



119875 009 006








Variable 1198601198731198831198601 1198711198661198601198711198781

CG GA CG GAGender



119875 063 004lowast 075 010H pylori



119875 047 061 024 073Genotype cagA













Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(a)


Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(b)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus15

minus10

(c)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

1210

86420


(d)

Mea

ns o

f va

lues

of

rela

tive

expr

essio

n (lo

g2)

10

75

5

25

0CG GA

ANXA1LGALS1

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200

250

150

100

50

0CGC GA

AnxA1

lowast

lowastlowast

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200 Gal-1

100

0CGC GA

lowast

lowastlowast

(e)





5 Conclusions


Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



























2(25mmolL) 20 120583L









119879) of






Status CGN ()

GAN ()






3 Results









4 Discussion




Variable 1198601198731198831198601 1198711198661198601198711198781




Variable 1198601198731198831198601 1198711198661198601198711198781



119875 015 039Smoking



119875 093 099Histology



119875 009 006








Variable 1198601198731198831198601 1198711198661198601198711198781

CG GA CG GAGender



119875 063 004lowast 075 010H pylori



119875 047 061 024 073Genotype cagA













Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(a)


Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(b)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus15

minus10

(c)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

1210

86420


(d)

Mea

ns o

f va

lues

of

rela

tive

expr

essio

n (lo

g2)

10

75

5

25

0CG GA

ANXA1LGALS1

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200

250

150

100

50

0CGC GA

AnxA1

lowast

lowastlowast

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200 Gal-1

100

0CGC GA

lowast

lowastlowast

(e)





5 Conclusions


Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Status CGN ()

GAN ()






3 Results









4 Discussion




Variable 1198601198731198831198601 1198711198661198601198711198781




Variable 1198601198731198831198601 1198711198661198601198711198781



119875 015 039Smoking



119875 093 099Histology



119875 009 006








Variable 1198601198731198831198601 1198711198661198601198711198781

CG GA CG GAGender



119875 063 004lowast 075 010H pylori



119875 047 061 024 073Genotype cagA













Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(a)


Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(b)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus15

minus10

(c)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

1210

86420


(d)

Mea

ns o

f va

lues

of

rela

tive

expr

essio

n (lo

g2)

10

75

5

25

0CG GA

ANXA1LGALS1

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200

250

150

100

50

0CGC GA

AnxA1

lowast

lowastlowast

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200 Gal-1

100

0CGC GA

lowast

lowastlowast

(e)





5 Conclusions


Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Variable 1198601198731198831198601 1198711198661198601198711198781




Variable 1198601198731198831198601 1198711198661198601198711198781



119875 015 039Smoking



119875 093 099Histology



119875 009 006








Variable 1198601198731198831198601 1198711198661198601198711198781

CG GA CG GAGender



119875 063 004lowast 075 010H pylori



119875 047 061 024 073Genotype cagA













Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(a)


Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(b)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus15

minus10

(c)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

1210

86420


(d)

Mea

ns o

f va

lues

of

rela

tive

expr

essio

n (lo

g2)

10

75

5

25

0CG GA

ANXA1LGALS1

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200

250

150

100

50

0CGC GA

AnxA1

lowast

lowastlowast

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200 Gal-1

100

0CGC GA

lowast

lowastlowast

(e)





5 Conclusions


Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Variable 1198601198731198831198601 1198711198661198601198711198781

CG GA CG GAGender



119875 063 004lowast 075 010H pylori



119875 047 061 024 073Genotype cagA













Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(a)


Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(b)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus15

minus10

(c)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

1210

86420


(d)

Mea

ns o

f va

lues

of

rela

tive

expr

essio

n (lo

g2)

10

75

5

25

0CG GA

ANXA1LGALS1

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200

250

150

100

50

0CGC GA

AnxA1

lowast

lowastlowast

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200 Gal-1

100

0CGC GA

lowast

lowastlowast

(e)





5 Conclusions


Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(a)


Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus10

ANXA1

(b)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

5

10

15

0

minus5

minus15

minus10

(c)


LGALS1

Valu

es o

f re

lativ

e ex

pres

sion

(log2

)

1210

86420


(d)

Mea

ns o

f va

lues

of

rela

tive

expr

essio

n (lo

g2)

10

75

5

25

0CG GA

ANXA1LGALS1

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200

250

150

100

50

0CGC GA

AnxA1

lowast

lowastlowast

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200 Gal-1

100

0CGC GA

lowast

lowastlowast

(e)





5 Conclusions


Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200

250

150

100

50

0CGC GA

AnxA1

lowast

lowastlowast

(e)






(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200 Gal-1

100

0CGC GA

lowast

lowastlowast

(e)





5 Conclusions


Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)

Mea

n op

tical

den

sitom

etry

(au

)

200 Gal-1

100

0CGC GA

lowast

lowastlowast

(e)





5 Conclusions


Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















5 Conclusions


Acknowledgment


References




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Expression of Annexin-A1 and Galectin-1 ...downloads.hindawi.com/journals/mi/2013/152860.pdf · Expression of Annexin-A1 and Galectin-1 Anti-Inflammatory Proteins