research article proinflammatory and anti-inflammatory...

Research ArticleProinflammatory and Anti-Inflammatory Cytokines Mediated byNF-120581B Factor as Prognostic Markers in Mammary Tumors

Gustavo Rodrigues Martins1 Gabriela Bottaro Gelaleti2 Marina Gobbe Moschetta1

Larissa Bazela Maschio-Signorini2 and Debora Ap Pires de Campos Zuccari3

1Laboratorio de Investigacao Molecular no Cancer (LIMC) Faculdade de Medicina de Sao Jose do Rio Preto (FAMERP)Department of Molecular Biology Graduate Program in Health Science Avenida Brigadeiro Faria Lima 5416 Vila Sao Pedro15090-000 Sao Jose do Rio Preto SP Brazil2Laboratorio de Investigacao Molecular no Cancer (LIMC) Faculdade de Medicina de Sao Jose do Rio Preto (FAMERP)Department of Biology Graduate Program in Genetics Universidade Estadual Paulista ldquoJulio de Mesquita Filhordquo (UNESPIBILCE)Rua Cristovao Colombo 2265 Jardim Nazareth 15054-000 Sao Jose do Rio Preto SP Brazil3Laboratorio de Investigacao Molecular no Cancer (LIMC) Faculdade de Medicina de Sao Jose do Rio Preto (FAMERP)Avenida Brigadeiro Faria Lima 5416 Vila Sao Pedro 15090-000 Sao Jose do Rio Preto SP Brazil

Correspondence should be addressed to Debora Ap Pires de Campos Zuccari deborazuccarifamerpbr

Received 26 October 2015 Revised 14 January 2016 Accepted 19 January 2016

Academic Editor Anshu Agrawal

Copyright copy 2016 Gustavo Rodrigues Martins et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Inflammation results in the production of cytokines such as interleukin- (IL-) 4 and IL-10 with immunosuppressive properties orIL-6 and TNF-120572 with procarcinogenic activity Furthermore NF-120581B is the major link between inflammation and tumorigenesisThis study verified the interaction between active inflammatory cytokines in the tumor microenvironment and serum of femaledogs withmammary tumors and their correlation with the clinicopathological characteristics and overall survival Measurement ofgene expression was performed by qPCR and protein levels by ELISALuminex High gene and protein expression levels of NF-120581BIL-6 and TNF-120572 were found in association with characteristics that reflect worse prognosis and a negative correlation betweenTNF-120572 protein expression and survival time was observed (119901 lt 005) In contrast high gene and protein expression levels of IL-4and IL-10were associatedwith characteristics of better prognosis and an increased level of IL-4 and a longer survival time of animalswere obtained (119901 lt 005) In addition there was a positive correlation between TNF-120572 and IL-6 expression in association with NF-120581B The results show a significant correlation of these cytokines with tumor development associated with NF-120581B expression andcytokines promodulation showing that these biological factors could be used as predictive and prognostic markers in breast cancer

1 Introduction

The microenvironment is a dynamic system that is largelycomposed of inflammatory cells and cytokines includingtumor cells and stroma (the latter of which consists ofimmune cells and the surrounding extracellular matrix) [12] Under specific stimulation conditions inflammation canresult in the production of antiangiogenic cytokines whichsuppress tumor growth Despite being antagonistic to theaction of tumor suppression in most tumors inflammatorycells can promote angiogenesis growth tumor cellmigration

tissue degradation and remodeling steps that are characteris-tic of the chronic inflammation associated with intense tissuedisorders typical of acute inflammation [1 3ndash7]

The transcription factor nuclear kappa B (NF-120581B) plays amajor role in innate immunity and inflammatory responses[8] and it is considered the major link between inflammationand tumorigenesis [1] This factor activates antiapoptoticgenes [9ndash11] and could promote the transcription of genesassociated with growth invasion and metastasis being thekey factor that allows precancerous and malignant cells toescape apoptosis [1]

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 9512743 10 pageshttpdxdoiorg10115520169512743

2 Mediators of Inflammation

Interleukins (ILs) could stimulate many signaling path-ways and thus regulate the transcription of target genesinvolved in cellular proliferation and survival [12] Besidesthat they act as immunemediators [13] and have been linkedto cancer playing a key role in the recruitment and position-ing of leukocytes in carcinogenesis [14] The recruitment andactivation of immune cells and the antitumor effects of IL-4an anti-inflammatory cytokine are involved in the inhibitionof angiogenesis [15] inflammation thrombosis growth andinvasion in some cancers [16 17] In addition to these effectson stromal cells some studies have reported that IL-4 hasantitumor functions by inducing apoptosis in several typesof tumor cells including breast cancer renal cell carcinomaand hepatocellular carcinoma [18]

IL-10 is a cytokine produced by Th1 and Th2 cells [19]as well as by tumor cells and has potent immunosuppressiveproperties [20] Originally known as an inhibitor factor of thesynthesis of cytokines [7] IL-10 suppresses the productionof IL-1120572 IL-1120573 tumor necrosis factor- (TNF-) 120572 IL-6 IL-8 IL-12 IL-18 granulocytes inflammatory macrophagesand NF-120581B [7 21 22] This cytokine acts concurrently withIL-3 and IL-4 to stimulate the proliferation of mast cellsperipheral blood lymphocytes [7] and macrophages andtheir anti-inflammatory action has been suggested in tumormicroenvironment [22]

On the other hand IL-6 has strong procarcinogenicactivity due to its role in tumor cell proliferation sur-vival angiogenesis inflammation and metastasis [7 23]In addition IL-6 is one of the activation signals for NF-120581B promoting cell differentiation and subsequent metastasis[24] thus reinforcing the close link between inflammatorymediators and neoplastic progression [1] Therefore TNF-120572 induces proinflammatory cytokines such as IL-1 [25] andIL-6 [26 27] during the inflammatory response [26ndash28]The interaction of this cytokine with its receptor (TNFR)induces apoptotic and antiapoptotic effects in different cellsystems [11] and it can promote cell proliferation and inhibitsapoptosis in the tumor microenvironment [29] at leastindirectly through the induction of NF-120581B [26]

A better understanding of the complexity of these bio-logical factorsrsquo network together with the characterizationof new genes aids in uncovering the molecular mechanismsof inflammatory diseases and cancer [30] In this way theobjective of this work was to ascertain the role of anti-inflammatory cytokines IL-4 and IL-10 and proinflamma-tory cytokines IL-6 and TNF-120572 in mammary tumors offemale dogs and to correlate these results with the gene andprotein expression of NF-120581B In addition we sought to verifythe correlation of these cytokines with clinicopathologicalparameters cancer progression and overall survival to deter-mine their prognostic value in mammary tumors

2 Material and Methods

21 Sample Processing Peripheral blood and tumor sampleswere obtained from30 female dogswithmalignantmammaryneoplasia (test group) attended to in Sao Jose do Rio PretoSP Brazil veterinary clinics in the years 2011 and 2012Animals with mammary tumors were included regardless

of breed weight or age These animals were followed upfor 540 days after tumor excision and blood collection Forcontrol group peripheral blood samples from 30 female dogsand five samples of mammary tissue adjacent to mammarycancer were obtained from animals that underwent electivehysterectomy as a preventive measure after completion ofthe consent form by their owners The criteria for the controlgroup were rigorously followed and included animals outsidethe estrus period animals with no tumor history and animalswith no detectable diseaseinflammation in the period orsurgery in the next study period In addition animals thatdied of causes other than tumor recurrence ormetastasis wereexcluded from the study

Peripheral blood (3mL) was collected in a CORVACserum separator tube (Labor Import Sao Paulo SP)stored at 4∘C processed by centrifugation (1000timesg 25min)and cryopreserved at minus80∘C for posterior Enzyme LinkedImmunosorbent Assay (ELISA) and xMAP protein analysisTumor samples were collected and divided into two piecesThe first one was fixed in 10 formaldehyde buffer for 24hours and paraffin embedded for histopathology classifica-tion after hematoxylin-and-eosin (HE) stain Tumor gradingwas ascertained according to Misdorp et al [31] by theArmed Forces Institute of Pathology (AFIP) The secondpiece was collected in a Falcon tube containing the RNA-stabilizing solution RNAlater (Life Technologiesreg) stored atroom temperature for 24 hours and posteriorly used for RNAextraction and qPCR analysis

22 Clinicopathological Characteristics Female dogs fromthe test group were evaluated with respect to physical (age)pathological (time course (interval between tumor diagnosisand surgical removal) tumor nodules location lymph nodeinvolvement tumor mass size clinical staging ulcerationand vascularization) and clinical (metastasis local recur-rence and death) characteristics (Table 1) The parametersemployed for the classification of clinical tumor grading werein accordance with the TNM (size lymph node involvementand metastasis) system established by the World HealthOrganization (WHO) for canine mammary gland tumors[32] The clinical grade was assigned as I II III or IVaccording to the extent of the tumor and the prognosis

The age of the animals varied from 7 to 14 years (mean =10 years) and there was predominance of Poodle (19) andDachshund (24) races In terms of the clinicopathologicalcharacteristics most animals had a tumor for more than sixmonths (54) and were of clinical stage III or IV (54)The proportions of animals with lymph node involvementanimals with metastasislocal recurrence or animals thatdied during the study were 16 27 and 27 respectively(Table 1)

23 Gene Expression by Quantitative RT-PCR (qPCR) Fortotal RNA extraction the tumor samples were manuallyprocessed into smaller pieces (100mgpiece) and the pieceswere then immersed in TRIzol reagent (Invitrogen LifeTechnologiesreg) andmacerated with a Politron (Dremel Mex-ico) Previously tumor samples were treated with DNase and10x buffer (10mMTris-HCl pH75 50mMCaCl

2 and 10mM

Mediators of Inflammation 3

Table 1 Clinicopathological parameters of female dogs with mam-mary tumors

Clinicopathological parameters Number of dogsAge of animalsgt10 years 14 (53)le10 years 16 (47)

Tumor time coursegtsix months 16 (54)lesix months 14 (44)

Tumor locationMultiple 14 (44)Single 16 (54)

VascularizationModerate 20 (66)Abundant 10 (34)

Tumor mass sizeT1 15 (50)T2T3 15 (50)

Lymph node involvementN0 25 (84)N1N2 5 (16)

MetastasisYes 8 (27)No 22 (73)

Clinical stagingI and II 14 (44)III and IV 16 (54)

RecurrenceYes 8 (27)No 22 (73)

CensorshipDeath 8 (27)Alive 22 (73)

MgCl2) (RNeasy Mini Kit Qiagen) followed by the addition

of ethylenediaminetetraacetic acid (EDTA) to deactivate oreliminate proteins binding to template DNA

The cDNA was synthesized using a High CapacitycDNA kit (Applied Biosystemsreg) The qPCR was per-formed in triplicate using the StepOne Plus Real TimePCR system (Applied Biosystems) and TaqMan Univer-sal Master Mix (Applied Biosystems) The following spe-cific primers were used NF-120581B Cf02622551 m1 (AppliedBiosystems) IL-4 Cf02623112 m1 (AppliedBiosystems) IL-6Cf02624153 m1 (Applied Biosystems) IL-10 Cf02624265 m1(Applied Biosystems) and TNF-120572 Cf02628236 m1 (AppliedBiosystems)

The following qPCR conditions were used an initial stepof 2 minutes at 50∘C denaturation for 10 minutes at 95∘C40 cycles at 95∘C for 15 seconds and 60∘C for one minute toanneal primers and extend the chains respectively and 35seconds at 65∘C to collect the signal The dissociation curvewas generated after amplification and included a step of 15seconds at 95∘C followed by 1min at 60∘C Each transcript

level was normalized to the expression of RPS19 (sense (51015840-GCC TTC CTC AAA AAG TCT GGG-31015840) antisense (51015840-GCT TGC TCC CTA CGA TGA GAA C-31015840) and probe(51015840-CCC TGA ATG GGT GGA C-31015840) (Applied Biosystems))andRPL8Cf02663820 m1 (Applied Biosystems) which wereused as endogenous control genes

The relative quantification (RQ) value of the expressionof interest genes was determined with DataAssist v30 bythe quantification method related to the average of thenormalizing genes used as endogenous controls (ΔΔCt) [33]and showed by Log

10The gene expression of each sample was

separately analyzed and classified as underexpressed (samplesgiving lt0 Log

10measurement) and overexpressed (samples

with quantification gt0 Log10) The samples were tested in

triplicate and all experiments included negative controls

24 Protein Expression by ELISA and xMAP Protein AssaysNF-120581B (dog nuclear factor (NF) kappa B p105 subunit (NF-120581B1) CUSABIO Biotech Co Hubei China) and IL-4 (USCNLife Science Inc Houston TX USA) protein expressionanalysis was performed according to the manufacturersrecommendations First of all the plate was precoated with aspecific antibody forNF-120581Band IL-4Then the standards andsamples were added in each well and incubated for 2 hoursat 37∘C A biotin-conjugated antibody and a horseradishperoxidase- (HRP-) avidin conjugate were added to theplate to amplify the signal and to increase the detectabilityof the target molecule A chromogenic substrate (3-3-5-5-tetramethylbenzidine TMB) was then added to detect theantibodyanalyte binding in the reaction Finally sulfuric acid(stop solution) was added to stabilize the color developmentto enable accurate measurement of the intensity at 450 nm

The analysis of IL-6 IL-10 and TNF-120572 proteins wasperformed using the MILLIPLEX MAP Kit (CCYTOMAG-90K Millipore Corporation USA) and the protein levelswere analyzed using the Luminex xMAPMAGPIX (MilliporeCorporation USA) The standard control and samples wereadded to the appropriate wells as were magnetic beads coatedwith specific antibodies for IL-6 IL-10 and TNF-120572 The platewas sealed and incubated overnight at 4∘Cwith agitation on aplate shakerThe detecting antibody was added and the platewas incubated Streptavidin-phycoerythrin was added toeach well to detect antibodyanalyte binding Measurementswere performed at 635 nm to excite the magnetic beads and525 nm to detect phycoerythrin

The reaction intensity was proportional to the concentra-tion of the cytokines and NF-120581B The calculation of OD wasdetermined through the adjustment curve four-parameterlogistic (4-PL) using the SkanIt software for theMultiskan FC251

25 Statistical Analysis The results were previously subjectedto descriptive analyses for the determination of normalityClinicopathological characteristics gene expression andpro-tein quantification were analyzed in the test and controlgroups and the differences between these groups weredetermined by Studentrsquos 119905-test or ANOVA followed by theBonferroni test All values are expressed as the mean plusmnstandard deviation (SD)


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Tumor location Vascularization Tumor size Metastasis Recurrence Clinical staging

minus20minus15minus10minus05

0005101520

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Tumorprogression

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(a)

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le6

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050

100150200250300350400

NF-120581

B (p

gm

L)

lowastlowastlowastlowast

lowastlowastlowast lowastlowastlowast

(b)

Figure 1NF-120581B gene overexpression correlated with (a) tumor progression greater than 6monthsmultiple tumor locations abundant tumorvascularization tumor mass size greater than 3 cm lymph node involvement presence of metastasis recurrence and clinical staging III orIV Higher NF-120581B protein levels correlated with (b) with age gt 10 years tumor progression greater than 6 months multiple tumor locationsabundant tumor vascularization and presence of metastasis Error bars plusmn standard error lowast119901 lt 005 lowastlowast119901 lt 001 lowastlowastlowast119901 lt 0001 indicatesignificant differences according to Studentrsquos 119905-test

The cutoff points for the protein analyses were establishedby the receiver operating characteristic (ROC) curve For theROC curve the protein expression levels from female dogsthat died were compared with those that survived until theend of the followup period Survival curves were plotted bythe Kaplan-Meier method and the differences between thecurves were evaluated by a log-rank test and hazard function

Spearmanrsquos rank correlation analysis was performed todescribe the correlation between proinflammatory and anti-inflammatory cytokines protein and gene expression withNF-120581B In addition the mutual relationship between proin-flammatory and anti-inflammatory cytokines was analyzedA multivariate logistic regression analysis was employed toevaluate the simultaneous influence of the prognostic factorson animal death A 119901 value lt005 was considered statisticallysignificant All analyses were performed using GraphPadPrism 4 and StatsDirect software

3 Results

31 Differential Gene and Protein Expression of NF-120581B IL-4 IL-10 IL-6 and TNF-120572 Associated with ClinicopathologicalFeatures and Survival The NF-120581B gene was significantlyoverexpressed in animalswith tumor time course greater thansix months (119901 lt 00001) multiple tumor locations (119901 =0005) abundant tumor vascularization (119901 = 0009) tumormass size greater than three centimeters (cm) (119901 = 0005)lymph node involvement (119901 = 0008) metastasis (119901 = 002)recurrence (119901 = 001) and clinical stage III or IV (119901 lt00001) (Figure 1(a))

In the same way the NF-120581B protein expression wassignificantly higher in animals older than 10 years (119901 =00001) as well as in animals with a tumor time course ofless than six months (119901 = 0001) multiple tumor locations(119901 = 001) abundant tumor vascularization (119901 = 003) andmetastasis (119901 = 00005) (Figure 1(b)) The best cutoff pointfor NF-120581B established by the ROC curve to discriminate thehigh risk of death was 2195 pgmL (sensitivity (95 CI) =71 specificity (95CI) = 41)No correlationwas observed

between NF-120581B serum levels and survival time (OR 200395 CI = 05553 to 8983 119901 = 029) (data not shown)

IL-4 was significantly overexpressed in animals aged le10 years (119901 = 002) and animals with tumor time courseof less than six months (119901 = 001) single tumor location(119901 = 00001) moderate tumor vascularization (119901 = 00002)tumor mass size less than three centimeters (119901 = 00006)no lymph node involvement (119901 = 0001) metastasis (119901 lt00001) recurrence (119901 = 00003) and clinical stage I or II(119901 lt 00001) and animals that were still alive at the end of thefollowup period (119901 lt 00001) (Figure 2(a))

Therefore IL-4 protein expression was significantlyhigher in animals aged le 10 years (119901 lt 00001) and animalswith tumor time course of less than six months (119901 = 004)moderate tumor vascularization (119901 = 003) tumor mass sizeof less than three centimeters (119901 = 0001) no metastasis(119901 = 001) no recurrence (119901 = 002) and clinical stage I or II(119901 = 0008) and animals that were still alive at the end of thefollowup period (119901 = 0005) (Figure 2(b)) Using the ROCcurve the best cutoff point for IL-4 to discriminate the highrisk of death was 3684 pgmL (sensitivity (95 CI) = 88specificity (95 CI) = 95) The Kaplan-Meier test demon-strated a positive correlation between IL-4 level and survivaltime (OR 0068184 95 CI = 0783029 to 1 119901 = 0007)(Figure 3)

For IL-10 significant gene overexpression was also cor-related with tumor time course of less than six months (119901 =0003) moderate tumor vascularization (119901 = 002) no lymphnode involvement (119901 = 00005) no metastasis (119901 = 001) norecurrence (119901 = 001) clinical stage I or II (119901 = 002) andanimals that were still alive at the end of the followup period(119901 = 0009) (Figure 4(a))

Likewise IL-10 protein expression was significantlyhigher in animals with tumor time course of less than sixmonths (119901 = 0003) single tumor location (119901 = 00009)moderate vascularization (119901 = 004) no recurrence (119901 =004) and clinical stage I or II (119901 = 003) and animals thatwere still alive during the followup (119901 = 004) (Figure 4(b))For the ROC curve for IL-10 the best cutoff point to


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live

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Age Vascularization Metastasis RecurrenceClinicalstaging Survival


001020

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Lymphnode

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progressionTumor

locationTumor

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lowastlowast lowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowastlowast

Log 1

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(a)

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nt T1T2

T3

No

Yes

No

Yes

I and

IIII

I and

IVA

live

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Age Vascularization Tumor size Metastasis Recurrence Clinical staging Survival

0100200300400500

IL-4

(pg

mL)

le6

mon

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gt6

mon

ths

le10

year

sgt10

year

s

Tumorprogression

lowastlowastlowastlowastlowastlowastlowastlowastlowast

lowastlowast lowastlowast

(b)

Figure 2 IL-4 gene overexpression correlated with (a) age le 10 years tumor progression for less than 6 months single tumor locationmoderate tumor vascularization tumor mass size less than 3 cm no lymph node involvement no metastasis no recurrence clinical stagingI or II and animals that were alive at the end of the followup period Higher IL-4 protein levels correlated with (b) with age le 10 years tumorprogression for less than 6 months moderate tumor vascularization tumor mass size less than 3 cm no metastasis no recurrence clinicalstaging I or II and animals that were alive at the end of the followup period Error bars plusmn standard error lowast119901 lt 005 lowastlowast119901 lt 001 lowastlowastlowast119901 lt 0001indicate significant differences according to Studentrsquos 119905-test

Surv

ival

()

0 100 200 300 400 500 600

DaysHigh expressionLow expression

0

20

40

60

80

100 Survival curvemdashIL-4

Figure 3 Survival curve of IL-4 Tumors of female dogs with highexpression (black line) of IL-4 had lower chance of death comparedwith animals with low expression (dotted line) 119901 = 0007

discriminate the high risk of death was 243 pgmL (sensitivity(95 CI) = 100 specificity (95 CI) = 53) No correlationwas observed between IL-10 protein expression and survivaltime (OR 03267 95 CI = 006811 to 1564 119901 = 016) (datanot shown)

In contrast IL-6 was significantly overexpressed in ani-mals aged gt 10 years (119901 = 003) and animals with multipletumor locations (119901 = 00005) abundant tumor vascular-ization (119901 = 0003) tumor mass size greater than threecentimeters (119901 = 001) lymph node involvement (119901 = 004)metastasis (119901 = 0003) recurrence (119901 = 003) and clinicalstage III or IV (119901 = 002) and animals that died duringfollowup (119901 = 0008) (Figure 5(a))

Mod

erat

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dant N0

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No

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No

Yes

I and

II

III a

nd IV

Aliv

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Tumor progression Vascularization Metastasis Recurrence Clinical staging Survival

le6

mon

ths

gt6

mon

ths


0005101520

Lymphnode

involvement

lowastlowast lowastlowastlowastlowastlowast lowastlowastlowastlowast

Log 1

0(R

Q)

(a)

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le

Mul

tiple

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Abun

dant No

Yes

I and

II

III a

nd IV

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Tumor progression Tumor location Vascularization Recurrence Clinical staging Survival

le6

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050

100150200250300350400

IL-1

0 (p

gm

L)

lowastlowastlowastlowastlowast

lowast

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(b)

Figure 4 IL-10 gene overexpression correlated with (a) tumorprogression for less than 6monthsmoderate tumor vascularizationno lymph node involvement no metastasis no recurrence clinicalstaging I or II and animals that were alive at the end of thefollowup period Higher IL-10 protein levels correlated with (b)tumor progression for less than 6 months single tumor locationmoderate tumor vascularization no recurrence clinical staging I orII animals that were alive at the end of the followup period Errorbars plusmn standard error lowast119901 lt 005 lowastlowast119901 lt 001 lowastlowastlowast119901 lt 0001 indicatesignificant differences according to Studentrsquos 119905-test

Similarly IL-6 protein expressionwas significantly higherin animals with tumor time course greater than six months(119901 = 0001) multiple tumor locations (119901 = 001) abundantvascularization (119901 = 002) tumor mass size greater than


Sing

leM

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dant T1

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nd II

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Aliv

eD

ead


le10

year

sgt10

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s


0005101520

Lymphnode

involvementTumor

location

lowastlowastlowastlowast lowastlowastlowastlowastlowastlowast lowast lowast lowast

lowast

Log 1

0(R

Q)

(a)

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Abun

dant T1

T2T

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Vascularization Tumor size Metastasis Recurrence SurvivalClinical staging

050

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IL-6

(pg

mL)

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Lymphnode

involvementTumor

location

lowastlowastlowastlowastlowastlowastlowastlowast


lowast lowast lowast

Tumorprogression

(b)

Figure 5 IL-6 gene overexpression correlatedwith (a) agele 10 yearsmultiple tumor locations abundant tumor vascularization tumormass size greater than 3 cm lymph node involvement metastasisrecurrence clinical staging III or IV and animals that died HigherIL-6 protein levels correlated with (b) tumor progression for morethan 6 months multiple tumor locations abundant tumor vascular-ization tumormass size greater than 3 cm lymphnode involvementmetastasis recurrence clinical staging III or IV and animals thatdied Error bars plusmn standard error lowast119901 lt 005 lowastlowast119901 lt 001 lowastlowastlowast119901 lt0001 indicate significant differences according to Studentrsquos 119905-test

three centimeters (119901 = 0001) lymph node involvement (119901 lt00001) metastasis (119901 lt 00001) recurrence (119901 = 0007)and clinical stage III or IV (119901 = 0001) and animals thatdied during followup (119901 = 004) (Figure 5(b))The best cutoffpoint for IL-6 established by the ROC curve to discriminatethe high risk of death was 7695 pgmL (sensitivity (95CI) = 75 specificity (95 CI) = 86) No correlation wasobserved between IL-6 protein expression and survival time(OR 1719 95 CI = 04045 to 9580 119901 = 043) (data notshown)

TNF-120572 was also overexpressed in animals with tumortime course greater than six months (119901 = 001) multipletumor locations (119901 = 0005) abundant tumor vascularization(119901 = 0007) tumor mass size greater than three centimeters(119901 = 002) metastasis (119901 = 0001) recurrence (119901 = 0003)and clinical stage III or IV (119901 = 00002) and animals that diedduring followup (119901 = 0005) (Figure 6(a))

The protein expression of TNF-120572was significantly higherin animals aged gt 10 years (119901 = 004) and animals with tumortime course greater than six months (119901 = 0004) abundantvascularization (119901 = 001) lymph node involvement (119901 =00007) metastasis (119901 = 002) and recurrence (119901 = 001) andanimals that died during followup (119901 = 004) (Figure 6(b))The best cutoff point to discriminate the high risk of deathestablished by the ROC curve was 163 pgmL (sensitivity(95 CI) = 87 specificity (95 CI) = 62) This analysis

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0005101520 SurvivalTumor location Vascularization Tumor size Metastasis Recurrence Clinical staging

Tumorprogression

lowastlowast lowastlowast lowastlowastlowastlowastlowastlowast lowastlowastlowastlowast lowast

Log 1

0(R

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(a)

Mod

erat

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Aliv

e

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le10

year

s

gt10

year

s

TNF-120572

(pg

mL)

0

50

100

150 Age Vascularization Metastasis Recurrence SurvivalTumor

progression

lowastlowast

lowastlowastlowast

lowast lowast lowast lowastlowast

Lymphnode

involvement

(b)

Figure 6 TNF-120572 gene overexpression correlated with (a) tumorprogression for more than 6 months multiple tumor locationsabundant tumor vascularization tumor mass size greater than 3 cmmetastasis recurrence clinical staging III or IV and animals thatdied Higher TNF-120572 protein levels correlated with (b) age gt 10years tumor progression for more than 6 months abundant tumorvascularization lymph node involvement metastasis recurrenceand animals that died (119901 = 004) Error bars plusmn standard errorlowast

119901 lt 005 lowastlowast119901 lt 001 lowastlowastlowast119901 lt 0001 indicate significant differencesaccording to Studentrsquos 119905-test

demonstrated a negative correlation between TNF-120572 proteinexpression and survival time (OR 5906 95 CI = 1387 to256 119901 = 001) (Figure 7)

32 Spearmanrsquos Rank Correlation Analysis between Proinflam-matory andAnti-Inflammatory Cytokines withNF-120581B Spear-manrsquos rank correlation demonstrated that proinflammatorycytokines IL-6 and TNF-120572 are mediated by NF-120581BThere wasa positive correlation between the proinflammatory TNF-120572 protein and gene expression when compared to NF-120581Bprotein and gene expression (119903 = 05233 119901 = 0003 119903 =06645 119901 lt 00001 Figures 8(a) and 8(b) resp) In the sameway there was a positive correlation between IL-6 and NF-120581B gene expression (119903 = 04012 119901 = 002) (Figure 8(b))Furthermore the mutual balance between proinflammatorycytokines and anti-inflammatory oneswas analyzed by Spear-manrsquos rank correlation and showed no correlation (119901 gt 005data not shown)

33 Multivariate Analysis The multivariate analysis of highlevels of the proinflammatory cytokines low levels of the anti-inflammatory cytokines and clinicopathological features ofpoor prognosis with regard to an increased risk of deathshowed no correlation (119901 gt 005) (Table 2)


Table 2 Multivariate analysis of proinflammatory and anti-inflammatory cytokines and clinicopathological features of poorprognosis with regard to an increased risk of death

Cytokines OD 95 CI 119901 valueIL-4 7819735 048417 to 126294945 01IL-6 13169 009401 to 18447342 08IL-10 0199689 001511 to 2639014 02TNF-120572 0259712 0011486 to 5872229 03

High expressionLow expression

0

20

40

60

80

100

Surv

ival

()

100 200 300 400 500 6000Days

TNF-120572

Figure 7 Survival curve of TNF-120572 Tumors of female dogs withhigh expression (dotted line) of TNF-120572 had higher chance of deathcompared with animals with low expression (black line) 119901 = 001

4 Discussion

NF-120581B is a crucial factor between chronic inflammation andcancer development [34] and it regulates both anti- andproinflammatory cytokines at various stages of tumorige-nesis and inflammation [35 36] We observed high geneand protein expression levels of NF-120581B in animals withtumor progression for greater than six months multipletumor locations abundant vascularization and metastasisindicating a role of this factor in angiogenesis supportand inhibition of apoptosis in tumor cells In accordancewith our prediction Kiliccioglu et al [37] observed thatNF-120581B inhibition through proteasome inhibition inducesapoptosis in human prostate cancer cell line (PC3) resultingin significantly increased protein levels of caspase-3 whichplays a central role in the execution phase of cell apoptosisSimilarly Chien et al [38] reported that pancreatic cancer cellgrowth is suppressed by inhibiting the NF-120581B pathway

In the same way high protein levels of NF-120581B wereobserved in female dogs above 10 years old which couldindicate the correlation between this nuclear factor andcell oxidative stress leading to tissue damage and chronicinflammation Corroborating with this a review of oxidative

IL-4IL-10

IL-6TNF-120572lowast

Correlation between NF-120581B and cytokines

NF-120581B protein expression

0

200

400

600

800

1000

Cyto

kine

s pro

tein

expr

essio

n

0 100 200 300 400 500

(a)

IL-4IL-10 TNF-120572lowast

IL-6lowast

4

3

2

21

minus2

minus2 minus1minus3

minus4

minus6

Cyto

kine

s gen

e exp

ress

ion

NF-120581B gene expression

(b)

Figure 8 Spearmanrsquos rank correlation analysis between proin-flammatory and anti-inflammatory cytokines with NF-120581B proteinand gene expression (a) positive correlation between NF-120581B pro-tein expression and the proinflammatory TNF-120572 cytokine proteinexpression (119903 = 05233 119901 = 0003) and (b) NF-120581B gene expressionand its correlation between the proinflammatory TNF-120572 cytokine(119903 = 06645 119901 lt 00001) and IL-6 (119903 = 04012 119901 = 002) geneexpression lowast119901 lt 005

stress and breast cancer recently published by Nourazarianet al [39] suggests that the increase of free radical levelsin tumor cells occurs under the influence of the increasedexpression of cellular enzymes and enhanced activity of somedependent tumor cells such as cancer-associated fibroblasts(CAFs) and tumor-associated macrophages (TAMs) [39 40]

Once NF-120581B has been activated through one of manypathways crucial properties of the malignant phenotype areactivated promoting tumor cell adaptation to the tumorenvironment [41] then we also reported NF-120581B overex-pressed in female dogs with tumor masses greater thanthree centimeters lymph node involvement recurrence andclinical stage III or IV leading to a poor survival rate of theanimals


It is known that NF-120581B is required for the expressionof many cytokine genes that encode proteins associatedwith inflammatorymediators [42]Then through Spearmanrsquosrank correlation we showed that high expression of NF-120581Bmodulates positively the IL-6 and TNF-120572 expression levelsin mammary tumors The proinflammatory cytokines IL-1120573IL-6 and TNF-120572 could be modulated by lipopolysaccharides(LPS) administration as it occurs in inflammatory diseasesAccording to Cheon et al [43] some drugs as saxifragininhibited the LPS-induced nuclear translocation of p65 andthe activation of caspase-1 in RAW 264 cells suggestingthat the NF-120581B-regulated gene transcription is responsiblefor anti-inflammatory response of macrophages In additionOsnes et al [44] affirmed that the inhibitory synthesis ofTNF-120572 by drugs as acetylsalicylic acid and sodium salicylatein human monocytes is due to interfering of nuclear translo-cation of NF-120581Bc-Rel proteins

Besides that corroborating with our study Al-Halabi etal [34] showed that the inhibition of NF-120581B significantlylowers microvessel density (CD31) and mRNA expressionlevels of IL-6 TNF-120572 and IL-1120572 as well as protein levels ofproliferationmarker (Ki-67) and vascular endothelial growthfactor- (VEGF-) A in human colon cancer confirming theprimordial role of NF-120581B in angiogenesis and thereforetumor progression

Regarding anti-inflammatory interleukins our study sus-tains a protector action of the anti-inflammatory cytokinesIL-4 and IL-10 in female dogsrsquo mammary tumors IL-4 washighly expressed in gene and protein levels in female dogswith a better prognostic This cytokine acts on endothelialcells inhibiting tumor-induced vascularization and starvingtumor cells [45] and also induces secretion of IL-10 the Th2cytokine with a strong suppressive effect on tumors [46 47]which could explain high levels of this cytokine in animalsthat are still alive after the followup period and in overallsurvival Furthermore according to Okada and Kuwashima[45] the sustained expression of IL-4 may provide effectivemeans for therapy of a variety of diseases including cancer

In addition high gene and protein expression of IL-10 were correlated with characteristics of better prognosisIL-10 downregulates proinflammatory cytokine expression[48 49] and inhibits the expression of CD31 [50] Lin andKarin [48] reported that IL-10 could modulate apoptosis andsuppress angiogenesis during tumor regression downregu-lating VEGF TNF-120572 and IL-6 production by TAMs Jindaland Borges [51] showed that IL-10 inhibits tumor growthby preventing chemokine expression and angiogenesis whenadministered at higher doses in cancer animalmodels Li et al[52] correlated high IL-10 expression in female dogs with nometastasis or recurrence andhigh overall survival Contrarilygene overexpression of IL-10 in animals that had metastasiscould suggest posttranscriptional regulation of this cytokineonce the protein levels do not show this correlation

IL-6mediates a plethora of physiological functions inclu-ding the developmental differentiation of lymphocytes cellproliferation and cell survival during tumorigenesis [48 53]This cytokine had high gene and protein expression corre-lated with poor prognosis and a negative correlation was

found in animals that died during the followup In the sameway a study by Madeddu et al [54] demonstrated highexpression of IL-6 through activation of AktPI3KmTORcascade due to elevated oxidative stress promoting onco-genesis and tumor progression Owing to its associationwith poor prognosis researchers have proposed IL-6 as atherapeutic target in cancer [49] Several phase III clinicaltrials are currently evaluating antibodies against IL-6 orIL-6R as therapeutic alternatives for prostate cancer [5355] and renal cancer [56 57] These results suggest thatfurther studies are needed to determine the appropriate useof anti-interleukin monoclonal antibodies as a therapeutictreatment and studies similar to ours evaluating interleukinexpression may be useful as a foundation for further clinicaltrials of therapy using cytokines

TNF-120572 is a cytokine that promotes tumorigenesis underconditions of unresolved inflammation [49] Increasing linesof evidence suggest that TNF-120572 regulates many of the criticalprocesses of tumor promotion and progression [48 58] Inour study high protein levels of TNF-120572were found in animalsthat died during the followup and correlatedwithworse prog-nostic features Its cytokine has been implicated yet as a tumorpromoter in different tumor types like ovarian cancer [59]gallbladder cancer [60] and oral squamous cell carcinoma[61] TNF-120572 binds to two receptors namely the ubiquitouslyexpressed TNF receptor 1 (TNFR1) and hematopoieticallyrestricted TNFR2 and modulates a signaling cascade thatinduces mediators of transcriptional regulation that are keyto cell survival invasion angiogenesis and impairment ofimmune surveillance in tumor biology [49 58 59 62]

Altogether our data showed the effect of cytokines ontumor development and progression as well as positivemodulation of NF-120581B and proinflammatory interleukins inmammary tumors Analysis of the active genes and proteinsin cancer can be used to target treatment more specificallyand our results suggest the use of these biological factors asprognostic biomarkers in cancer

5 Conclusion

Our results are of utmost importance for a better understand-ing of the dynamic network of cytokines and the influence ofNF-120581B on tumorigenesisThese cytokines could be employedas noninvasive prognostic markers in female dogs withmammary tumors and could be useful for predicting diseaseprogression and tumor recurrence There is lack of studiesdevoted to diagnostic therapeutic and prognostic markersin canine mammary tumors so this study defines potentialprognostic and predictive markers for routine use in theclinic allowing the detection of recurrence and metastasiswhich could allow the early adoption ofmore precise conductto improve the overall survival of animals

Ethical Approval

This study was approved by the Ethics Committee of the SaoJose do Rio Preto Medical School (Protocol no 39452009)


Conflict of Interests

The authors declare no commercial or financial conflict ofinterests

Acknowledgments

The authors wish to acknowledge Fundacao de Amparoa Pesquisa do Estado de Sao Paulo (FAPESP) Brazil forresearch funding (201206098-0) and CAPES for Ms stu-dentship And they also wish to thank the Clinical Veteri-narians in Sao Jose do Rio Preto and region that promptlycollaborated with the research

References

[1] G Lorusso and C Ruegg ldquoThe tumor microenvironment andits contribution to tumor evolution toward metastasisrdquo Histo-chemistry and Cell Biology vol 130 no 6 pp 1091ndash1103 2008

[2] G C Leonardi S Candido M Cervello et al ldquoThe tumormicroenvironment in hepatocellular carcinoma (review)rdquo Inter-national Journal of Oncology vol 40 no 6 pp 1733ndash1747 2012

[3] R I Tepper R L Coffman and P Leder ldquoAn eosinophil-dependent mechanism for the antitumor effect of interleukin-4rdquo Science vol 257 no 5069 pp 548ndash551 1992

[4] C Lu C Sheehan J W Rak C A Chambers N Hozumiand R S Kerbel ldquoEndogenous interleukin 6 can function asan in vivo growth-stimulatory factor for advanced-stage humanmelanoma cellsrdquoClinical Cancer Research vol 2 no 8 pp 1417ndash1425 1996

[5] M Kobayashi H Kobayashi R B Pollard and F Suzuki ldquoApathogenic role of Th2 cells and their cytokine products onthe pulmonary metastasis of murine B16 melanomardquo Journal ofImmunology vol 160 no 12 pp 5869ndash5873 1998

[6] F Benchetrit A Ciree V Vives et al ldquoInterleukin-17 inhibitstumor cell growth bymeans of a T-cell-dependent mechanismrdquoBlood vol 99 no 6 pp 2114ndash2121 2002

[7] Hamidullah B Changkija and R Konwar ldquoRole of interleukin-10 in breast cancerrdquo Breast Cancer Research and Treatment vol133 no 1 pp 11ndash21 2012

[8] M Karin ldquoNuclear factor-120581B in cancer development andprogressionrdquo Nature vol 441 no 7092 pp 431ndash436 2006

[9] W E Naugler and M Karin ldquoNF-120581B and cancer-identifyingtargets and mechanismsrdquo Current Opinion in Genetics andDevelopment vol 18 no 1 pp 19ndash26 2008

[10] A A Beg and D Baltimore ldquoAn essential role for NF-120581B inpreventing TNF-120572-induced cell deathrdquo Science vol 274 no5288 pp 782ndash784 1996

[11] D K Biswas K J Martin C McAlister et al ldquoApoptosiscaused by chemotherapeutic inhibition of nuclear factor-120581Bactivationrdquo Cancer Research vol 63 no 2 pp 290ndash295 2003

[12] G B Gelaleti B V Jardim C Leonel M G Moschetta and DA P D C Zuccari ldquoInterleukin-8 as a prognostic serummarkerin canine mammary gland neoplasiasrdquo Veterinary Immunologyand Immunopathology vol 146 no 2 pp 106ndash112 2012

[13] J R Wilczynski ldquoCancer and pregnancy share similar mecha-nisms of immunological escaperdquo Chemotherapy vol 52 no 3pp 107ndash110 2006

[14] A Nicolini A Carpi andG Rossi ldquoCytokines in breast cancerrdquoCytokine and Growth Factor Reviews vol 17 no 5 pp 325ndash3372006

[15] M Saleh I D Davis and A F Wilks ldquoThe paracrine roleof tumour-derived mIL-4 on tumour-associated endotheliumrdquoInternational Journal of Cancer vol 72 no 4 pp 664ndash672 1997

[16] J L Gooch A V Lee andD Yee ldquoInterleukin 4 inhibits growthand induces apoptosis in human breast cancer cellsrdquo CancerResearch vol 58 no 18 pp 4199ndash4205 1998

[17] MGomesACoelhoAAraujo A L Teixeira R Catarino andR Medeiros ldquoInfluence of functional genetic polymorphism (minus590CT) in non-small cell lung cancer (NSCLC) developmentthe paradoxal role of IL-4rdquoGene vol 504 no 1 pp 111ndash115 2012

[18] J L Gooch B Christy and D Yee ldquoSTAT6 mediatesinterleukin-4 growth inhibition in human breast cancer cellsrdquoNeoplasia vol 4 no 4 pp 324ndash331 2002

[19] A OrsquoGarra and P Vieira ldquoTH1 cells control themselves byproducing interleukin-10rdquo Nature Reviews Immunology vol 7no 6 pp 425ndash428 2007

[20] M C Heckel A Wolfson C A Slachta et al ldquoHuman breasttumor cells express IL-10 and IL-12p40 transcripts and proteinsbut do not produce IL-12p70rdquo Cellular Immunology vol 266no 2 pp 143ndash153 2011

[21] A B Lentsch T P Shanley V Sarma and P A Ward ldquoIn vivosuppression of NF-kappa B and preservation of I kappa B alphaby interleukin-10 and interleukin-13rdquo The Journal of ClinicalInvestigation vol 100 no 10 pp 2443ndash2448 1997

[22] J B Mumm J Emmerich X Zhang et al ldquoIL-10 elicits IFN120574-dependent tumor immune surveillancerdquo Cancer Cell vol 20no 6 pp 781ndash796 2011

[23] D Drygin C B Ho M Omori et al ldquoProtein kinase CK2modulates IL-6 expression in inflammatory breast cancerrdquoBiochemical and Biophysical Research Communications vol 415no 1 pp 163ndash167 2011

[24] G Germano P Allavena andAMantovani ldquoCytokines as a keycomponent of cancer-related inflammationrdquo Cytokine vol 43no 3 pp 374ndash379 2008

[25] M-A Le Bitoux and I Stamenkovic ldquoTumor-host interactionsthe role of inflammationrdquo Histochemistry and Cell Biology vol130 no 6 pp 1079ndash1090 2008

[26] S Kim T O Keku C Martin et al ldquoCirculating levels of infla-mmatory cytokines and risk of colorectal adenomasrdquo CancerResearch vol 68 no 1 pp 323ndash328 2008

[27] G Soria M Ofri-Shahak I Haas et al ldquoInflammatory media-tors in breast cancer coordinated expression of TNF120572amp IL-1120573with CCL2 amp CCL5 and effects on epithelial-to-mesenchymaltransitionrdquo BMC Cancer vol 11 article 130 2011

[28] S Madhusudan S R Muthuramalingam J P Braybrooke et alldquoStudy of etanercept a tumor necrosis factor-alpha inhibitor inrecurrent ovarian cancerrdquo Journal of Clinical Oncology vol 23no 25 pp 5950ndash5959 2005

[29] F Tang G Tang J Xiang QDaiM R Rosner andA Lin ldquoTheabsence of NF-120581B-mediated inhibition of c-Jun N-terminalkinase activation contributes to tumor necrosis factor alpha-induced apoptosisrdquoMolecular and Cellular Biology vol 22 no24 pp 8571ndash8579 2002

[30] M Kawaguchi M Adachi N Oda F Kokubu and S-KHuang ldquoIL-17 cytokine familyrdquo Journal of Allergy and ClinicalImmunology vol 114 no 6 pp 1265ndash1273 2004

[31] W Misdorp R W Else E Hellmen and E Lipscomb ldquoDefini-tions and explanatory notesrdquo inWho Histological Classificationof Mammary Tumors of the Dog and Cat pp 18ndash27 ArmedForces Institute of Pathology Washington DC USA 1999


[32] G D Cassali A C Bertagnolli G E LavalleW L F Tavares EFerreira and A E Silva ldquoPerspectives for diagnosis prognosisand treatment of mammary neoplasms in dogsrdquo in Proceedingsof the 34th World Small Animal Veterinary Congress (WSAVArsquo09) Sao Paulo Brazil 2009

[33] T D Schmittgen and K J Livak ldquoAnalyzing real-time PCR databy the comparative CT methodrdquo Nature Protocols vol 3 no 6pp 1101ndash1108 2008

[34] R Al-Halabi M Bou Chedid R Abou Merhi et al ldquoGallotan-nin inhibits NF120581B signaling and growth of human colon cancerxenograftsrdquoCancer Biology andTherapy vol 12 no 1 pp 59ndash682011

[35] S C Gupta C Sundaram S Reuter and B B AggarwalldquoInhibiting NF-120581B activation by small molecules as a therapeu-tic strategyrdquo Biochimica et Biophysica Acta vol 1799 no 10ndash12pp 775ndash787 2010

[36] A Del Prete P Allavena G Santoro R Fumarulo M MCorsi and A Mantovani ldquoMolecular pathways in cancer-related inflammationrdquoBiochemiaMedica vol 21 no 3 pp 264ndash275 2011

[37] I Kiliccioglu E Konac N Varol S Gurocak and C YucelBilen ldquoApoptotic effects of proteasome and histone deacetylaseinhibitors in prostate cancer cell linesrdquo Genetics and MolecularResearch vol 13 no 2 pp 3721ndash3731 2014

[38] W Chien D H Lee Y Zheng et al ldquoGrowth inhibitionof pancreatic cancer cells by histone deacetylase inhibitorbelinostat through suppression of multiple pathways includingHIF NFkB andmTOR signaling in vitro and in vivordquoMolecularCarcinogenesis vol 53 no 9 pp 722ndash735 2014

[39] A R Nourazarian P Kangari and A Salmaninejad ldquoRoles ofoxidative stress in the development and progression of breastcancerrdquo Asian Pacific Journal of Cancer Prevention vol 15 no12 pp 4745ndash4751 2014

[40] V Sosa T Moline R Somoza R Paciucci H Kondoh and ME LLeonart ldquoOxidative stress and cancer an overviewrdquo AgeingResearch Reviews vol 12 no 1 pp 376ndash390 2013

[41] M Tafani B Pucci A Russo et al ldquoModulators of HIF1120572and NFkB in cancer treatment is it a rational approach forcontrolling malignant progressionrdquo Frontiers in Pharmacologyvol 4 article 13 2013

[42] A S Baldwin ldquoThe transcription factor NF-120581B and humandiseaserdquo Journal of Clinical Investigation vol 1 pp 3ndash6 2001

[43] S Y Cheon K S Chung E Jeon A Nugroho H J Parkand H J An ldquoAnti-inflammatory activity of saxifragin viainhibition of NF-120581B involves caspase-1 activationrdquo Journal ofNatural Products vol 78 no 7 pp 1579ndash1585 2015

[44] L T N Osnes K B Foss G B Joo et al ldquoAcetylsalicylic acidand sodium salicylate inhibit LPS-induced NF-kappa Bc-Relnuclear translocation and synthesis of tissue factor (TF) andtumor necrosis factor alfa (TNF-alpha) in human monocytesrdquoThrombosis and Haemostasis vol 76 no 6 pp 970ndash976 1996

[45] H Okada and N Kuwashima ldquoGene therapy and biologictherapy with interleukin-4rdquo Current Gene Therapy vol 2 no4 pp 437ndash450 2002

[46] Z Li L Chen and Z Qin ldquoParadoxical roles of IL-4 in tumorimmunityrdquoCellular andMolecular Immunology vol 6 no 6 pp415ndash422 2009

[47] F Pericle M Giovarelli M P Colombo et al ldquoAn effi-cient Th2-type memory follows CD8+ lymphocyte-driven andeosinophil-mediated rejection of a spontaneous mouse mam-mary adenocarcinoma engineered to release IL-4rdquo Journal ofImmunology vol 153 no 12 pp 5659ndash5673 1994

[48] W-W Lin and M Karin ldquoA cytokine-mediated link betweeninnate immunity inflammation and cancerrdquo Journal of ClinicalInvestigation vol 117 no 5 pp 1175ndash1183 2007

[49] G Landskron M De la Fuente P Thuwajit C Thuwajit andM A Hermoso ldquoChronic inflammation and cytokines in thetumor microenvironmentrdquo Journal of Immunology Researchvol 2014 Article ID 149185 19 pages 2014

[50] J Shi J Li H Guan et al ldquoAnti-fibrotic actions of interleukin-10 against hypertrophic scarring by activation of PI3KAKT andSTAT3 signaling pathways in scar-forming fibroblastsrdquo PLoSONE vol 9 no 5 Article ID e98228 2014

[51] S Jindal and V F Borges ldquoThe emerging role of cytokines inbreast cancer from initiation to survivorshiprdquo Breast Cancervol 23 pp 113ndash126 2011

[52] Y Li H Yu S Jiao and J Yang ldquoPrognostic value of IL-10expression in tumor tissues of breast cancer patientsrdquo Xi BaoYu Fen Zi Mian Yi Xue Za Zhi vol 30 no 5 pp 517ndash520 2014

[53] J Karkera H Steiner W Li et al ldquoThe anti-interleukin-6 antibody siltuximab down-regulates genes implicated intumorigenesis in prostate cancer patients from a phase I studyrdquoProstate vol 71 no 13 pp 1455ndash1465 2011

[54] C Madeddu G Gramignano C Floris G Murenu G Sollaiand A Maccio ldquoRole of inflammation and oxidative stress inpost-menopausal oestrogen-dependent breast cancerrdquo Journalof Cellular andMolecularMedicine vol 18 no 12 pp 2519ndash25292014

[55] K Fizazi J S De Bono A Flechon et al ldquoRandomised phaseII study of siltuximab (CNTO 328) an anti-IL-6 monoclonalantibody in combination with mitoxantroneprednisone ver-sus mitoxantroneprednisone alone in metastatic castration-resistant prostate cancerrdquo European Journal of Cancer vol 48no 1 pp 85ndash93 2012

[56] T Puchalski U Prabhakar Q Jiao B Berns and H M DavisldquoPharmacokinetic and pharmacodynamic modeling of an anti-interleukin-6 chimeric monoclonal antibody (siltuximab) inpatients with metastatic renal cell carcinomardquo Clinical CancerResearch vol 16 no 5 pp 1652ndash1661 2010

[57] J-F Rossi S Negrier N D James et al ldquoA phase III studyof siltuximab (CNTO 328) an anti-interleukin-6 monoclonalantibody in metastatic renal cell cancerrdquo British Journal ofCancer vol 103 no 8 pp 1154ndash1162 2010

[58] P Szlosarek K A Charles and F R Balkwill ldquoTumour necrosisfactor-120572 as a tumour promoterrdquoEuropean Journal of Cancer vol42 no 6 pp 745ndash750 2006

[59] K A Charles H Kulbe R Soper et al ldquoThe tumor-promotingactions of TNF-120572 involve TNFR1 and IL-17 in ovarian cancer inmice and humansrdquo Journal of Clinical Investigation vol 119 no10 pp 3011ndash3023 2009

[60] G Zhu Q Du X Wang N Tang F She and Y ChenldquoTNF-120572 promotes gallbladder cancer cell growth and invasionthrough autocrinemechanismsrdquo International Journal ofMolec-ular Medicine vol 33 no 6 pp 1431ndash1440 2014

[61] S H Lee H S Hong Z X Liu et al ldquoTNF120572 enhances cancerstem cell-like phenotype via Notch-Hes1 activation in oralsquamous cell carcinoma cellsrdquo Biochemical and BiophysicalResearch Communications vol 424 no 1 pp 58ndash64 2012

[62] F Balkwill ldquoTNF-120572 in promotion and progression of cancerrdquoCancer andMetastasis Reviews vol 25 no 3 pp 409ndash416 2006

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


Interleukins (ILs) could stimulate many signaling path-ways and thus regulate the transcription of target genesinvolved in cellular proliferation and survival [12] Besidesthat they act as immunemediators [13] and have been linkedto cancer playing a key role in the recruitment and position-ing of leukocytes in carcinogenesis [14] The recruitment andactivation of immune cells and the antitumor effects of IL-4an anti-inflammatory cytokine are involved in the inhibitionof angiogenesis [15] inflammation thrombosis growth andinvasion in some cancers [16 17] In addition to these effectson stromal cells some studies have reported that IL-4 hasantitumor functions by inducing apoptosis in several typesof tumor cells including breast cancer renal cell carcinomaand hepatocellular carcinoma [18]

IL-10 is a cytokine produced by Th1 and Th2 cells [19]as well as by tumor cells and has potent immunosuppressiveproperties [20] Originally known as an inhibitor factor of thesynthesis of cytokines [7] IL-10 suppresses the productionof IL-1120572 IL-1120573 tumor necrosis factor- (TNF-) 120572 IL-6 IL-8 IL-12 IL-18 granulocytes inflammatory macrophagesand NF-120581B [7 21 22] This cytokine acts concurrently withIL-3 and IL-4 to stimulate the proliferation of mast cellsperipheral blood lymphocytes [7] and macrophages andtheir anti-inflammatory action has been suggested in tumormicroenvironment [22]

On the other hand IL-6 has strong procarcinogenicactivity due to its role in tumor cell proliferation sur-vival angiogenesis inflammation and metastasis [7 23]In addition IL-6 is one of the activation signals for NF-120581B promoting cell differentiation and subsequent metastasis[24] thus reinforcing the close link between inflammatorymediators and neoplastic progression [1] Therefore TNF-120572 induces proinflammatory cytokines such as IL-1 [25] andIL-6 [26 27] during the inflammatory response [26ndash28]The interaction of this cytokine with its receptor (TNFR)induces apoptotic and antiapoptotic effects in different cellsystems [11] and it can promote cell proliferation and inhibitsapoptosis in the tumor microenvironment [29] at leastindirectly through the induction of NF-120581B [26]

A better understanding of the complexity of these bio-logical factorsrsquo network together with the characterizationof new genes aids in uncovering the molecular mechanismsof inflammatory diseases and cancer [30] In this way theobjective of this work was to ascertain the role of anti-inflammatory cytokines IL-4 and IL-10 and proinflamma-tory cytokines IL-6 and TNF-120572 in mammary tumors offemale dogs and to correlate these results with the gene andprotein expression of NF-120581B In addition we sought to verifythe correlation of these cytokines with clinicopathologicalparameters cancer progression and overall survival to deter-mine their prognostic value in mammary tumors

2 Material and Methods

21 Sample Processing Peripheral blood and tumor sampleswere obtained from30 female dogswithmalignantmammaryneoplasia (test group) attended to in Sao Jose do Rio PretoSP Brazil veterinary clinics in the years 2011 and 2012Animals with mammary tumors were included regardless

of breed weight or age These animals were followed upfor 540 days after tumor excision and blood collection Forcontrol group peripheral blood samples from 30 female dogsand five samples of mammary tissue adjacent to mammarycancer were obtained from animals that underwent electivehysterectomy as a preventive measure after completion ofthe consent form by their owners The criteria for the controlgroup were rigorously followed and included animals outsidethe estrus period animals with no tumor history and animalswith no detectable diseaseinflammation in the period orsurgery in the next study period In addition animals thatdied of causes other than tumor recurrence ormetastasis wereexcluded from the study

Peripheral blood (3mL) was collected in a CORVACserum separator tube (Labor Import Sao Paulo SP)stored at 4∘C processed by centrifugation (1000timesg 25min)and cryopreserved at minus80∘C for posterior Enzyme LinkedImmunosorbent Assay (ELISA) and xMAP protein analysisTumor samples were collected and divided into two piecesThe first one was fixed in 10 formaldehyde buffer for 24hours and paraffin embedded for histopathology classifica-tion after hematoxylin-and-eosin (HE) stain Tumor gradingwas ascertained according to Misdorp et al [31] by theArmed Forces Institute of Pathology (AFIP) The secondpiece was collected in a Falcon tube containing the RNA-stabilizing solution RNAlater (Life Technologiesreg) stored atroom temperature for 24 hours and posteriorly used for RNAextraction and qPCR analysis

22 Clinicopathological Characteristics Female dogs fromthe test group were evaluated with respect to physical (age)pathological (time course (interval between tumor diagnosisand surgical removal) tumor nodules location lymph nodeinvolvement tumor mass size clinical staging ulcerationand vascularization) and clinical (metastasis local recur-rence and death) characteristics (Table 1) The parametersemployed for the classification of clinical tumor grading werein accordance with the TNM (size lymph node involvementand metastasis) system established by the World HealthOrganization (WHO) for canine mammary gland tumors[32] The clinical grade was assigned as I II III or IVaccording to the extent of the tumor and the prognosis

The age of the animals varied from 7 to 14 years (mean =10 years) and there was predominance of Poodle (19) andDachshund (24) races In terms of the clinicopathologicalcharacteristics most animals had a tumor for more than sixmonths (54) and were of clinical stage III or IV (54)The proportions of animals with lymph node involvementanimals with metastasislocal recurrence or animals thatdied during the study were 16 27 and 27 respectively(Table 1)

23 Gene Expression by Quantitative RT-PCR (qPCR) Fortotal RNA extraction the tumor samples were manuallyprocessed into smaller pieces (100mgpiece) and the pieceswere then immersed in TRIzol reagent (Invitrogen LifeTechnologiesreg) andmacerated with a Politron (Dremel Mex-ico) Previously tumor samples were treated with DNase and10x buffer (10mMTris-HCl pH75 50mMCaCl

2 and 10mM





























Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2N

oYe

sN

oYe

sI a

nd II

III a

nd IV



0005101520

le6

mon

ths

gt6

mon

ths

Tumorprogression



Lymphnode

involvement

Log 1

0(R

Q)

(a)

Sing

le

Mul

tiple

Mod

erat

e

Abun

dant No

Yes


le6

mon

ths

gt6

mon

ths

le10

year

s

gt10

year

s

050

100150200250300350400

NF-120581

B (p

gm

L)



(b)




3 Results









Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2

No

Yes

No

Yes

I and

IIII

I and

IVA

live

Dea

d



001020

le6

mon

ths

gt6

mon

ths

le10

year

sgt10

year

s

Lymphnode

involvementTumor

progressionTumor

locationTumor

size


Log 1

0(R

Q)

(a)

Mod

erat

eAb

unda

nt T1T2

T3

No

Yes

No

Yes

I and

IIII

I and

IVA

live

Dea

d


0100200300400500

IL-4

(pg

mL)

le6

mon

ths

gt6

mon

ths

le10

year

sgt10

year

s

Tumorprogression



(b)


Surv

ival

()

0 100 200 300 400 500 600


0

20

40

60

80





Mod

erat

e

Abun

dant N0

N1

N2

No

Yes

No

Yes

I and

II

III a

nd IV

Aliv

e

Dea

d


le6

mon

ths

gt6

mon

ths


0005101520

Lymphnode

involvement


Log 1

0(R

Q)

(a)

Sing

le

Mul

tiple

Mod

erat

e

Abun

dant No

Yes

I and

II

III a

nd IV

Aliv

e

Dea

d


le6

mon

ths

gt6

mon

ths

050

100150200250300350400

IL-1

0 (p

gm

L)


lowast

lowast

lowastlowast

(b)




Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2N

oYe

sN

oYe

sI a

nd II

III a

nd IV

Aliv

eD

ead


le10

year

sgt10

year

s


0005101520

Lymphnode

involvementTumor

location


lowast

Log 1

0(R

Q)

(a)

Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2N

oYe

sN

oYe

sI a

nd II

III a

nd IV

Aliv

eD

ead


050

100150200250

IL-6

(pg

mL)

le6

mon

ths

gt6

mon

ths

Lymphnode

involvementTumor

location




Tumorprogression

(b)





Sing

leM

ultip

leM

oder

ate

Abun

dant No

Yes

No

Yes

I and

IIII

I and

IVA

live

Dea

d

le6

mon

ths

gt6

mon

ths

T1T2

T3



Tumorprogression


Log 1

0(R

Q)

(a)

Mod

erat

e

Abun

dant

le6

mon

ths

gt6

mon

ths

N0

N1

N2

No

Yes

No

Yes

Aliv

e

Dea

d

le10

year

s

gt10

year

s

TNF-120572

(pg

mL)

0

50

100


progression

lowastlowast

lowastlowastlowast


Lymphnode

involvement

(b)










0

20

40

60

80

100

Surv

ival

()

100 200 300 400 500 6000Days

TNF-120572


4 Discussion



IL-4IL-10




0

200

400

600

800

1000

Cyto

kine

s pro

tein

expr

essio

n

0 100 200 300 400 500

(a)


IL-6lowast

4

3

2

21

minus2

minus2 minus1minus3

minus4

minus6

Cyto

kine

s gen

e exp

ress

ion


(b)













5 Conclusion


Ethical Approval





Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2N

oYe

sN

oYe

sI a

nd II

III a

nd IV



0005101520

le6

mon

ths

gt6

mon

ths

Tumorprogression



Lymphnode

involvement

Log 1

0(R

Q)

(a)

Sing

le

Mul

tiple

Mod

erat

e

Abun

dant No

Yes


le6

mon

ths

gt6

mon

ths

le10

year

s

gt10

year

s

050

100150200250300350400

NF-120581

B (p

gm

L)



(b)




3 Results









Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2

No

Yes

No

Yes

I and

IIII

I and

IVA

live

Dea

d



001020

le6

mon

ths

gt6

mon

ths

le10

year

sgt10

year

s

Lymphnode

involvementTumor

progressionTumor

locationTumor

size


Log 1

0(R

Q)

(a)

Mod

erat

eAb

unda

nt T1T2

T3

No

Yes

No

Yes

I and

IIII

I and

IVA

live

Dea

d


0100200300400500

IL-4

(pg

mL)

le6

mon

ths

gt6

mon

ths

le10

year

sgt10

year

s

Tumorprogression



(b)


Surv

ival

()

0 100 200 300 400 500 600


0

20

40

60

80





Mod

erat

e

Abun

dant N0

N1

N2

No

Yes

No

Yes

I and

II

III a

nd IV

Aliv

e

Dea

d


le6

mon

ths

gt6

mon

ths


0005101520

Lymphnode

involvement


Log 1

0(R

Q)

(a)

Sing

le

Mul

tiple

Mod

erat

e

Abun

dant No

Yes

I and

II

III a

nd IV

Aliv

e

Dea

d


le6

mon

ths

gt6

mon

ths

050

100150200250300350400

IL-1

0 (p

gm

L)


lowast

lowast

lowastlowast

(b)




Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2N

oYe

sN

oYe

sI a

nd II

III a

nd IV

Aliv

eD

ead


le10

year

sgt10

year

s


0005101520

Lymphnode

involvementTumor

location


lowast

Log 1

0(R

Q)

(a)

Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2N

oYe

sN

oYe

sI a

nd II

III a

nd IV

Aliv

eD

ead


050

100150200250

IL-6

(pg

mL)

le6

mon

ths

gt6

mon

ths

Lymphnode

involvementTumor

location




Tumorprogression

(b)





Sing

leM

ultip

leM

oder

ate

Abun

dant No

Yes

No

Yes

I and

IIII

I and

IVA

live

Dea

d

le6

mon

ths

gt6

mon

ths

T1T2

T3



Tumorprogression


Log 1

0(R

Q)

(a)

Mod

erat

e

Abun

dant

le6

mon

ths

gt6

mon

ths

N0

N1

N2

No

Yes

No

Yes

Aliv

e

Dea

d

le10

year

s

gt10

year

s

TNF-120572

(pg

mL)

0

50

100


progression

lowastlowast

lowastlowastlowast


Lymphnode

involvement

(b)










0

20

40

60

80

100

Surv

ival

()

100 200 300 400 500 6000Days

TNF-120572


4 Discussion



IL-4IL-10




0

200

400

600

800

1000

Cyto

kine

s pro

tein

expr

essio

n

0 100 200 300 400 500

(a)


IL-6lowast

4

3

2

21

minus2

minus2 minus1minus3

minus4

minus6

Cyto

kine

s gen

e exp

ress

ion


(b)













5 Conclusion


Ethical Approval





Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2

No

Yes

No

Yes

I and

IIII

I and

IVA

live

Dea

d



001020

le6

mon

ths

gt6

mon

ths

le10

year

sgt10

year

s

Lymphnode

involvementTumor

progressionTumor

locationTumor

size


Log 1

0(R

Q)

(a)

Mod

erat

eAb

unda

nt T1T2

T3

No

Yes

No

Yes

I and

IIII

I and

IVA

live

Dea

d


0100200300400500

IL-4

(pg

mL)

le6

mon

ths

gt6

mon

ths

le10

year

sgt10

year

s

Tumorprogression



(b)


Surv

ival

()

0 100 200 300 400 500 600


0

20

40

60

80





Mod

erat

e

Abun

dant N0

N1

N2

No

Yes

No

Yes

I and

II

III a

nd IV

Aliv

e

Dea

d


le6

mon

ths

gt6

mon

ths


0005101520

Lymphnode

involvement


Log 1

0(R

Q)

(a)

Sing

le

Mul

tiple

Mod

erat

e

Abun

dant No

Yes

I and

II

III a

nd IV

Aliv

e

Dea

d


le6

mon

ths

gt6

mon

ths

050

100150200250300350400

IL-1

0 (p

gm

L)


lowast

lowast

lowastlowast

(b)




Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2N

oYe

sN

oYe

sI a

nd II

III a

nd IV

Aliv

eD

ead


le10

year

sgt10

year

s


0005101520

Lymphnode

involvementTumor

location


lowast

Log 1

0(R

Q)

(a)

Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2N

oYe

sN

oYe

sI a

nd II

III a

nd IV

Aliv

eD

ead


050

100150200250

IL-6

(pg

mL)

le6

mon

ths

gt6

mon

ths

Lymphnode

involvementTumor

location




Tumorprogression

(b)





Sing

leM

ultip

leM

oder

ate

Abun

dant No

Yes

No

Yes

I and

IIII

I and

IVA

live

Dea

d

le6

mon

ths

gt6

mon

ths

T1T2

T3



Tumorprogression


Log 1

0(R

Q)

(a)

Mod

erat

e

Abun

dant

le6

mon

ths

gt6

mon

ths

N0

N1

N2

No

Yes

No

Yes

Aliv

e

Dea

d

le10

year

s

gt10

year

s

TNF-120572

(pg

mL)

0

50

100


progression

lowastlowast

lowastlowastlowast


Lymphnode

involvement

(b)










0

20

40

60

80

100

Surv

ival

()

100 200 300 400 500 6000Days

TNF-120572


4 Discussion



IL-4IL-10




0

200

400

600

800

1000

Cyto

kine

s pro

tein

expr

essio

n

0 100 200 300 400 500

(a)


IL-6lowast

4

3

2

21

minus2

minus2 minus1minus3

minus4

minus6

Cyto

kine

s gen

e exp

ress

ion


(b)













5 Conclusion


Ethical Approval





Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2N

oYe

sN

oYe

sI a

nd II

III a

nd IV

Aliv

eD

ead


le10

year

sgt10

year

s


0005101520

Lymphnode

involvementTumor

location


lowast

Log 1

0(R

Q)

(a)

Sing

leM

ultip

leM

oder

ate

Abun

dant T1

T2T

3N

0N

1N

2N

oYe

sN

oYe

sI a

nd II

III a

nd IV

Aliv

eD

ead


050

100150200250

IL-6

(pg

mL)

le6

mon

ths

gt6

mon

ths

Lymphnode

involvementTumor

location




Tumorprogression

(b)





Sing

leM

ultip

leM

oder

ate

Abun

dant No

Yes

No

Yes

I and

IIII

I and

IVA

live

Dea

d

le6

mon

ths

gt6

mon

ths

T1T2

T3



Tumorprogression


Log 1

0(R

Q)

(a)

Mod

erat

e

Abun

dant

le6

mon

ths

gt6

mon

ths

N0

N1

N2

No

Yes

No

Yes

Aliv

e

Dea

d

le10

year

s

gt10

year

s

TNF-120572

(pg

mL)

0

50

100


progression

lowastlowast

lowastlowastlowast


Lymphnode

involvement

(b)










0

20

40

60

80

100

Surv

ival

()

100 200 300 400 500 6000Days

TNF-120572


4 Discussion



IL-4IL-10




0

200

400

600

800

1000

Cyto

kine

s pro

tein

expr

essio

n

0 100 200 300 400 500

(a)


IL-6lowast

4

3

2

21

minus2

minus2 minus1minus3

minus4

minus6

Cyto

kine

s gen

e exp

ress

ion


(b)













5 Conclusion


Ethical Approval





Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















0

20

40

60

80

100

Surv

ival

()

100 200 300 400 500 6000Days

TNF-120572


4 Discussion



IL-4IL-10




0

200

400

600

800

1000

Cyto

kine

s pro

tein

expr

essio

n

0 100 200 300 400 500

(a)


IL-6lowast

4

3

2

21

minus2

minus2 minus1minus3

minus4

minus6

Cyto

kine

s gen

e exp

ress

ion


(b)













5 Conclusion


Ethical Approval





Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

























5 Conclusion


Ethical Approval





Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















Acknowledgments


References





































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article proinflammatory and anti-inflammatory...

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