Research Article Effect of Vasoactive Intestinal Peptide (VIP) on … · 2019. 7. 31. · er NK cells were incubated with the VIP, its antagonist for h in -well plate as mentioned

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 429545 10 pageshttpdxdoiorg1011552013429545

Research ArticleEffect of Vasoactive Intestinal Peptide (VIP) on NKG2DSignal Pathway and Its Contribution to Immune Escape ofMKN45 Cells

Chong Wang Xi-Jin Zhou Yuan-yuan Li Juan Wan Le-ying Yang and Guo-Hua Li

Department of Gastroenterology The First Affiliated Hospital of Nanchang University Nanchang Jiangxi 330006 China

Correspondence should be addressed to Guo-Hua Li liguohua98sohucom

Received 24 June 2013 Accepted 13 August 2013

Academic Editors T Hida and K Higai

Copyright copy 2013 Chong Wang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective To investigate VIP effect on the cytotoxicity of NK cell to gastric cancer cells in vitro and the relation between the effectwith the NKG2D signal molecules in NK cellsMaterial and Methods NK cells were purified from peripheral blood mononuclearcells (PBMC) Before and after NK cells were incubated with VIP or its antagonist (D-p-Cl-Phe6Leu17)-VIP we detected thecytotoxicity ofNK cells toMKN45 gastric cancer cells byMTT and detected the expressions ofNKG2DDAP10 andNF-120581Bproteinsand mRNAs in NK cells by immunocytochemistry and RT-PCR in those conditions Then we analyzed the effect of VIP and itsantagonist on the cytotocicity of NK cell to gastric cancer cells and on expressions of NKG2D DAP10 and NF-120581B signal moleculesin NK cells Results VIP could inhibit the cytotoxicity of NK cells to MKN45 cells and could inhibit the expressions of NKG2DDAP10 and NF-120581B in NK cells However (D-p-Cl-Phe6 Leu17)-VIP could reverse those effects Conclusions The VIP inhibitedthe cytotoxicity of NK cell to MKN45 cells which might get through inhibiting the expressions of NKG2D signal molecules in NKcells This may be one mechanism of gastric cancer cells escaping organism immune clearance

1 Introduction

Gastric cancer is the most common malignancy in gastroin-testinal tract The occurrence of gastric cancer must escapeorganism immune surveillance in order to be cleaned Thenonspecific immune cells take important role in direct cyto-toxicity on tumor cells especially the natural killer (NK) cellsThe NK cells have a unique function that is killing tumorcells or spontaneously transformed carcinoma cells withoutprevious sensitization and MHC restriction [1] So NK cellsplay amajor role in the immune cleaningNatural killer group2memberD (NKG2D)which is the cellmembrane activatingreceptor of some kinds of immune cells (especially NK cells)plays a key role in its cytotoxicity [2] NKG2Dhas been shownto be important in the NK cell-mediated control of somecancers [3] Human NK cells only express the long isoformof NKG2D and it associates with DAP-10 to induce both acytotoxicity and cytokine-mediated immune response [4 5]

On the other hand it has been known that gastriccancer cells could escape organism immunosurveillance by

the following mechanisms [6] (i) the expressions of tumorantigens were absent or decreased on the surface of tumorcells (ii) the expressions ofmajor histocompatibility complex(MHC) class I molecules on the tumor cells were reduced toa very low level (iii) the tumor cells expressing FasL whichinduced the apoptosis of lymphocytes that expressed Fas(iv) the expressions of costimulatory signal molecules wereabsent in tumor cells and (v) tumor cells could secrete somecytokines or hormones such as VIP IL-10 which inhibitedorganism immune function

The gastrointestinal hormone vasoactive intestinal pep-tide (VIP) belongs to the secretinVIP family which wasinitially isolated from the intestine [7] and secreted byneurons endocrine cells immune cells [8 9] and gastriccarcinoma cells [10] VIP affect not only gastrointestinal func-tions but also organism immune functions through bindingto G protein-coupled receptors [11] which are distributedin most human tissues [12] Many studies showed that VIPenhanced Th2 cells respond inhibited Th1 cell proliferation[13ndash15] impacted B cell differentiation [16 17] and inhibited

2 The Scientific World Journal

NK cell activity So VIP is the most important immuneinhibiting neuropeptide Many studies showed that VIP wasrelated with the gastric cancer Hejna et al reported that theserum VIP concentration increased in patients with gastriccancer [18]We have showed that the gastric adenocarcinomatissues contained secreting VIP cancer cells [10] Above thissuggested that VIPmight play an important role in inhibitingorganism immune functions

So we suppose that VIP which is secreted by gastriccancer cells may facilitate gastric cancer cells to escapeorganism immune cleaning by inhibiting NKG2D signalmolecules in NK cells To confirm it we want to research theinfluence of VIP on NK cells cytotoxicity to kill the gastricadenocarcinoma cell line (MKN45 cells) in vitro and researchthe relationship of this influence with the NKG2D signalmolecules in NK cells

2 Materials and Methods

21 Natural Killer (NK) Cells Separation Purification andIdentification Heparinized bloodwas collected between 600and 1000 am from 10 healthy volunteers of the staff at theNanchang University (mean age 426 plusmn 84 60 male nohistory of chronic illness and without current prescriptionmedications usage) The peripheral blood mononuclear cells(PBMC) were obtained by standard Ficoll-Hypaque den-sity gradient centrifugation and suspended by RPMI-1640media with 10 heat-inactivated fetal bovine serum (FBS)(Gibco Invitrogen Grand Island NY USA) The activatedNK cells were purified from PBMC by complement lysis(CDC) method [19] and cultured in RPMI-1640 mediumwith 10 newborn calf serum (NBCS) (Gibco InvitrogenGrand Island NY USA) 1 times 106UL recombinant humaninterleukin-2 (rhIL-2 PeproTech Rocky Hill NJ USA) and10mgL phytohaemagglutinin (PHA Sigma St Louis MOUSA) under a microaerophilic environment (at 37∘C 2O2 5 CO

2) The NK cells (expressed CD3minusCD16+CD56+)

percentage was detected by FACS after incubated with anti-human CD3 FITC(CD16+CD56) PE Cocktail (BioLegendSan Diego CA USA) according to its instruction

22 Cell Culture The MKN45 cell line originated fromhuman poorly differentiated gastric adenocarcinoma (fromATCC company) The cells were cultured in RPMI-1640medium with 10 NBCS under a microaerophilic environ-ment (at 37∘C 2 O

2 5 CO

2)

23 The Cytotoxicity of NK Cells on the Growth of MKN45Cells The MKN45 cells were cultured in the medium ina 96-well plate Each well contained 200 120583L cancer cellssolution in a 1 times 104 cellsmL concentration The supernatewas thrown away after the MKN45 cells were cultured for4 h then each well was added NK cells andor different drugsolutions for different incubated time according to differentgroups as follows Each plate included 4 wells for the negativecontrols (seeded byMKN45 andmedium) and 4 wells for theblank controls (only seeded by 200 120583L medium) Each group

included 4 wells at least in each time Each test repeated threetimes

231 Effect of VIP on the Growth of NK Cells to MKN45 CellsIn this test the MKN45 cells in 96-well plate were added180 120583L NK cells solution in a 1 times 105 cellsmL concentration(the ratio of effect to target was 10 1) and 20120583L VIP solutionsto each well for 48 h The final concentration of VIP in eachwell was 1 times 10minus5molL to 1 times 10minus7molL respectively

232 Interaction between VIP and Its Antagonist In thistest the MKN45 cells in 96-well plate were added 180 120583LNK cells solution in a 1 times 105 cellsmL concentration and20120583L different drug solutions to each well for 48 h The drugsolution included VIP its antagonist ([D-p-Cl-Phe6 Leu17]-VIP Sigma St Louis MO USA) or VIP combined with itsantagonist The final concentration of VIP in each well was 1times 10minus6molL its antagonist in each well was 1 times 10minus4 1 times 10minus5or 1 times 10minus6molL respectively

233 MTT The growth state of MKN45 cells in eachtest mentioned above were measured by methyl thiazolyldiphenyl tetrazolium bromide assay (MTT) The OD value(OD490

) in MTT represented the quantity of living MKN45cells but not NK cells because the suspending NK cells werethrown away in the course of removing supernate duringMTT test

24 Effect of VIP or Its Antagonist on NKG2D SignalMoleculesof NK Cells NK cells were cultured in the medium in a 6-well plate Each well contained 2970 120583L NK cells solutionin a 1 times 106 cellsmL and 30120583L different drug solutions toeach well The drug solution included VIP its antagonistor mixed solution of VIP and its antagonist The finalconcentration of VIP and its antagonist in each well was10minus6molL and 10minus5molL respectively After incubation for48 h the NK cells were used to detect target gene mRNA andprotein expressions by RT-PCR or immunocytochemistrytest Each group included 3 wells at least in each test (negativecontrol group contained 2970 120583L NK cells solution in a 1times 106 cellsmL concentration and 30120583L medium) The testrepeated three times

241 RNA Isolation and Reverse Transcription PolymeraseChain Reaction (RT-PCR)

RNA Isolation After NK cells were incubated by the VIP andits antagonist for 48 h as mentioned above the total RNA ofthe NK cells was extracted according to the procedure of theTrizol reagent (TianGen Biotechnology Co Beijing China)

RT-PCR A typical 25-120583L reaction contained 10120583L totalRNA 200 units of Moloney murine leukemia virus (MMLV)reverse transcriptase 500 ng oligo(dT)

15 1 times 5 120583L reverse

transcription buffer 10mmolL deoxy-ribonucleoside tri-phosphate (dNTP) RNasin inhibitor (Promega Madison

The Scientific World Journal 3

WI USA) and ddH2OThe reaction was incubated for 1 h at

42∘C and then heated at 95∘C for 5min to inactivate the reac-tionThe cDNAwas used as a template for PCR amplificationThe PCR for each cDNA was performed in a 25-120583L volumeincluding 2120583L template 2timesTaq PCR MasterMix (TianGenBiotechnology Co Beijing China) 5120583L 03120583molL each oftarget gene primers 1 and 2 and ddH

2O under the following

conditions 95∘C for 5min followed by 35 cycles at 94∘C for30 s 55∘C for 30 s (51∘C for NKG2D andVPAC1 52∘C for VIPandNF-120581B 54∘C forDAP10) and 72∘C for 1min finally 5minat 72∘CThe primers are listed in Table 1

Electrophoresis andmRNA SemiquantityThe 4 120583L PCR prod-uct of each target mRNA was added with 1120583L bromophenolblue buffer andwas electrophoresed for 30min on agarose gelstained with ethidium bromide (05mgmL) The length ofthe PCR products of VIP VPAC1 NKG2D DAP10 NF-120581Band beta-actin was 361 bp 432 bp 365 bp 282 bp 326 bp and500 bp respectively The marker was DNAmarker DL600(TianGen Biotechnology Co Beijing China) We examinedand photographed each gel under UV light The integratedoptical density of each PCR product was measured by theHPIAS-1000 color picture analysis system (Olympus TokyoJapan) The ratio of integrated optical density of the targetgene to that of reference beta-actin was calculated whichrepresented the relative expression quantity of the target gene

242 Immunocytochemistry After NK cells were incubatedwith the VIP its antagonist for 48 h in 6-well plate asmentioned above the NK cells were collected and weresuspended to solution in 1 times 105mL concentration The cellsolution was dropped on slides and was fixed by 95 coldalcohol After slides dried the target proteins of NK cellswere detected by immunocytochemistry test according tothe procedure of the PV-9000 kit (for detecting VIP DAP10VPAC1 and NF-120581B) or PV-6003 kit (for detecting NKG2D)(Zhong Shan Company Beijing China) The primary anti-bodies of rabbit DAP10 polyclonal antibody rabbit VPAC1polyclonal antibodymouseNF-120581Bp65monoclonal antibodygoatNKG2Dpolyclonal antibody (SantaCruz BiotechnologySanta Cruz CA USA) and rabbit VIP polyclonal antibody(Abcam Cambridge UK) were diluted to 1 100 1 200 1 501 100 and 1 1000 with 001molL PBS respectively The testset negative controls by replacing respectively the primaryantibody with normal rabbit mice or goat serum underthe same experimental conditions The positive particle wasshown as dark brown under microscopy The percentage ofVIP DAP10 VPAC1 NF-120581B or NKG2D positive cells was theaverage percentage of positive cells in five randomly selectedfields under a 200x magnification microscopy

25 Statistical Analysis The statistical analysis was done byusing SPSS 120 for windows (Chicago IL USA) The resultswere reported as mean plusmn SD t-test was used to investigatethe differences between two sessions If ANOVA revealed asignificant difference among three or more sessions SNK-test was used to investigate the differences between any two

Table 1 List of primers

Beta-actin primer 1 (sense) 51015840CGCTACAGCTTCACCACCAC31015840

Beta-actin primer 2(antisense) 51015840TACTCCTGCTTGCTGATCCAC31015840

VIP primer 1 (sense) 51015840TCCTTGTGCTCCTGACTCTT31015840

VIP primer 2 (antisense) 51015840GACTGCATCTGAGTGACGTT31015840

VPAC1 primer 1 (sense) 51015840CTATGTGCAGATGATCGAGG31015840

VPAC1 primer 2 (antisense) 51015840GAAGAGGTGCATGTGGATGT31015840

NKG2D primer 1 (sense) 51015840TTCTGCTGCTTCATCGCTGT31015840

NKG2D primer 2(antisense) 51015840GGTGAGAGAATGGAGCCATC31015840

DAP10 primer 1 (sense) 51015840CAGTCCACCATGATCCATCT31015840

DAP10 primer 2 (antisense) 51015840TGCCTGGCATGTTGATGTAG31015840

NF-120581Bp65 primer 1 (sense) 51015840GAGAGGAGCACAGATACCAC31015840

NF-120581Bp65 primer 2(antisense) 51015840CACAGCATTCAGGTCGTAGT31015840

sessions Kruskal-Wallis 119867-test or Chi-square analysis wasused for the assessment of enumeration data

3 Results

31 NK Cells Separation Efficiency (Table 2) NK cells werepurified fromPBMCand identified by FACSThepurificationof NK cells could reach 60 by CDC method (Figure 1)

32 The Cytotoxic Effect of NK Cells on the Growth of MKN45Cells and VIP Effect on It (Table 3) When MKN45 cellssolution was incubated with different drugs andor NK cellssolution for 48 h we found that OD

490value of MKN45 cells

in only adding NK cells groups decreased more than that inaddingNK cells with differentVIP concentrations (119875 lt 005)The higher VIP concentration was themoreOD

490value was

1 times 10minus6molL VIP could obviously inhibit the cytotoxicity ofNK cells to MKN45 cells However there was no significantdifference in the OD

490values between only adding different

VIP groups with negative control group (119875 gt 005)

33 Interaction between VIP and Its Antagonist (Table 4)When MKN45 cells solution was incubated with NK cellssolution and 10minus6molL VIP combined with different antag-onist (1 times 10minus4molL 1 times 10minus5molL and 1 times 10minus6molL)for 48 h we found that the OD

490values in the groups in

presence of VIP and its antagonist were lower than that inthe groups in present of VIP (119875 lt 005) The more antagonistconcentration was the less the OD

490value was That is to

say 1 times 10minus5molL antagonist could abolish the VIP influencecompletelyHowever therewas no significant difference in theOD490

value between groups only added different antagonistwith negative control group (119875 gt 005)

34 VIP and Its Antagonist Influence on NK Cellsrsquo Cytotox-icity in the Different Time (Table 5) When MKN45 cellssolution was incubated with NK cells solution andor the 1times 10minus6molL VIP or 1 times 10minus5molL antagonist in wells for


100

101

102

103

104

100

101

102

103

104

FL1-height

FL2

-hei

ght

Quad Gated ()

UL

UR

LL

LR

018

012

9923

048

(a)

100

101

102

103

104

100

101

102

103

104

NK

CD3 FITC

6005

2385

890

720

Quad

UL

UR

LL

LR

Gated ()

(b)

100

101

102

103

104

100

101

102

103

104

NK

CD3 FITC

1527

1672

3498

3303

Quad

UL

UR

LL

LR

Gated ()

(c)

Figure 1 NK cells separation efficiency (a) negative control group (b) NK cells purity by CDC method and (c) NK cells purity in PBMC(UL represented CD3minusCD16++CD56+ NK cells)


Table 2 The comparison of NK cells purity between group A andB

Group n Purification of NK cells ()A 10 60583 119883

2= 36750

B 10 18508 119875 lt 001

Group A NK cells purity after purified from PBMC by CDCmethod GroupB NK cells purity in PBMC

Table 3The cytotoxicity of NK cells to MKN45 cells and VIP effecton it

Group n OD490

A MKN45 + NK + VIP (1 times 10minus5) 12 0161 plusmn 0024ab

B MKN45 + NK + VIP (1 times 10minus6) 12 0157 plusmn 0017ab

C MKN45 + NK + VIP (1 times 10minus7) 12 0138 plusmn 0022abc

D MKN45 + VIP (1 times 10minus5) 12 0196 plusmn 0019b

E MKN45 + VIP (1 times 10minus6) 12 0200 plusmn 0028b

F MKN45 + VIP (1 times 10minus7) 12 0201 plusmn 0025b

G MKN45 + NK 12 0106 plusmn 0016a

H MKN45 (blank control) 12 0209 plusmn 0026

119865 = 31533119875 lt 001 a stands for119875 lt 005 comparedwith groupH b standsfor 119875 lt 005 compared with group G c stands for 119875 lt 005 compared withgroup A

Table 4 The cytotoxicity of VIP in 10minus6 molL and its antagonist in10minus4 to 10minus6 molL concentration for 48 h on the growth of NK cellsto MKN45 cells

Group n OD490

A MKN45 + NK + VIP + antagonist(1 times 10minus4) 12 0114 plusmn 0017

acd

B MKN45 + NK + VIP + antagonist(1 times 10minus5) 12 0115 plusmn 0018

acd

C MKN45 + NK + VIP + antagonist(1 times 10minus6) 12 0141 plusmn 0025

abce

D MKN45 + antagonist (1 times 10minus4) 12 0195 plusmn 0024bde

E MKN45 + antagonist (1 times 10minus5) 12 0197 plusmn 0023bde

F MKN45 + antagonist (1 times 10minus6) 12 0194 plusmn 0024bde

G MKN45 + NK + VIP 12 0156 plusmn 0019abc

H MKN45 + VIP 12 0196 plusmn 0027b

I MKN45 + NK 12 0104 plusmn 0024a

J MKN45 (blank control) 12 0208 plusmn 0027

119865 = 36751 119875 lt 001 a stands for 119875 lt 005 compared with group J b standsfor 119875 lt 005 compared with group I c stands for 119875 lt 005 compared withgroupH d stands for119875 lt 005 compared with groupG e stands for119875 lt 005compared with group B

24 h 48 h and 72 h receptively we found that there was nosignificant difference in OD

490values between only adding

VIP or antagonist groups and control group from 24 h to 72 h(119875 gt 005) There was significant difference in OD

490values

between adding NK cells solution and control group from48 h to 72 h (119875 lt 005) but not 24 h (119875 gt 005) The OD

490

values in groups added VIP and NK cells were significantlyhigher than that in groups added only with NK cells in 48 h(119875 lt 005) but not in 24 h or 72 h (119875 gt 005) There wasno significant difference in OD

490values between the groups

added VIP its antagonist and NK cells and the groups onlyadded NK cells from 24 h to 72 h (119875 gt 005)

35The Expressions of NKG2D DAP10 andNF-120581B p65mRNAand Protein The expression of VIP mRNA and protein didnot find in NK cells andMKN45 cells however VPAC1 couldbe detected in two kinds of cells (Figures 2 and 3)

The expressions of NKG2D DAP10 and NF-120581B mRNAand protein in NK cells decreased when NK cells incubatedwith VIP (119875 lt 005 Table 6) VIP antagonist could partiallyor completely abolished the effect of VIP on the expressionsof NKG2D DAP10 and NF-120581B in NK cells (119875 lt 005 Table 6Figures 4 5 6 and 7)

4 Discussion

Gastric cancer was once the second most common cancerin the world In most undeveloped countries the mor-bidity of stomach cancer had increased over the past halfcentury In China stomach cancer was the most commonmalignant neoplasm The gastric cancer cells must escapefrom organism immunosurveillance which could clean thetransformed tumor cells In organism immunosurveillancesystem the nonspecific immune cells especially the NK cellswere the first line against tumor cells or virus infected cellsand played an important role in directly cytotoxic effecton tumor cells NK cells could kill tumor or spontaneousmetastatic carcinoma cells without previous sensitization orMHC restricted [1] In this study we observed that NKcells had indeed cytotoxicity on tumor cells (MKN45 akind of gastric adenocarcinoma cell line) However howcould gastric cancer cells escape from organism immuneclearance It was reported by the following mechanisms[6] (i) the expressions of tumor antigens were absent ordecreased on the surface of tumor cells (ii) the expressions ofmajor histocompatibility complex (MHC) class I moleculeson the tumor cells were reduced to a very low level (iii)the tumor cells expressed FasL which induced the apoptosisof lymphocytes that expressed Fas (iv) the expressions ofcostimulatory signal molecules were absent in tumor cellsand (v) tumor cells could produce cytokine or hormone suchas IL-10 which inhibited organism immune function

Many studies showed that VIP was related with thegastric cancer [10 18 20] We had showed that some gastriccancer cells in tumor tissues could secrete VIP [10] Thephysiological functions of VIP in the GI tract were associ-ated with the secretion and motor of the digestive systemRecently many studies showed that VIP inhibited immunefunction For example VIP enhanced respondence of Th2cells inhibited Th1 cell proliferation [13ndash15] impacted Bcell differentiation [16 17] and inhibited NK cell activitythrough several pathways So we wonder that if VIP whichwas secreted by gastric cancer cells facilitate gastric cancercells to escape immunosurveillance by inhibiting NK cellsactivity To confirm it we designed the study In our study weobserved that VIP could significantly inhibit the cytotoxicityof NK cells on MKN45 cells at the 10 1 ratio of effect to


Table 5 The cytotoxicity of VIP or antagonist in 1 times 10minus6 molL 1 times 10minus5 molL concentration respectively on the growth of NK cells toMKN45 cells for 24 h 48 h or 72 h

Time (h) n OD490

A B C D E F24 12 0115 plusmn 0320 0102 plusmn 0028 0124 plusmn 0031 0121 plusmn 0037 0093 plusmn 0029 0126 plusmn 0038

48 12 0115 plusmn 0021lowast0153 plusmn 0017

lowast0197 plusmn 0015

0200 plusmn 0028

0106 plusmn 0016


72 12 0319 plusmn 0100lowast0381 plusmn 0109 0375 plusmn 0097 0381 plusmn 0120 0346 plusmn 0124


lowast119875 lt 005 compared with group F in same time 119875 lt 005 compared with group E in same time Group A MKN45 + NK + VIP + antagonist group B

MKN45 + NK + VIP group C MKN45 + antagonist group D MKN45 + VIP group E MKN45 + NK and group F MKN45 (control)

(a)

(b)

Figure 2 VIP and VPAC1 protein expression in NK cells times200 ((a)showed VIP negative expression and (b) showed VPAC1 positiveexpression)

target and could be reversed completely by VIP antagonistIt confirmed our speculation

It has been reported that endogenous VIP affect thegrowth of some gastric cancer cells [10] by binding to G-protein-coupled receptors [11 21] We observed that exoge-nous VIP and VIP antagonist did not affect the proliferationof MKN45 cells So we excluded the influence of VIP orantagonist on MKN45 cell That is to say VIP and antagonistaffected OD

490of MKN45 by acting on NK cells

It has been know that the NK cells cytotoxicity wasthrough the activating receptor of NK cells NKG2D NKG2Dhad been shown to be important in the NK cell-mediatedcontrol of some cancersNKG2Dwas aC-type lectin-like typereceptor and belonged to theNKgroup 2 (NKG2) of receptorsas member D Although NKG2D belonged to the NKG2

M K C

600

500

400

300

200

(a)

M K C

(b)

M K C B A

(c)

Figure 3 Electrophoresis of PCR product of VIP VPAC1 and beta-actin mRNA in different groups (a) stands for VIP (b) for VPAC1and (c) for beta-actin M stands for marker DL600 K for MKN45cells C for NK cells A for NK cells + VIP and B for NK cells + VIP+ VIP antagonist The length of PCR products of VIP VPAC1 andbeta-actin was 361 bp 432 bp and 500 bp

family it did not share most of their properties In contrast toother members of the family NKG2D was a homodimer andrecognized a number of stressed cells induced by MHC classI-like ligands [22 23] NKG2D as a molecular sensor coulddetect ldquoinduced self rdquo on cells in danger which was mostlytriggered by viral infections and by some factors to causeDNA damage and tumor transformation [2] NKG2D signaltransduction should through two adaptor proteins DAP10and DAP12 [5] which were associated with the receptor ashomodimers DAP12 carried an immunoreceptor tyrosine-based activation motif (ITAM) [24] DAP10 had a YINMmotif in its cytoplasmatic tail [4 5] Human NK cells onlyexpressed the long isoform of NKG2D (NKG2D-L) and itwas associated with DAP-10 to induce immune response Itwas reported that NKG2D signal pathway might touch uponother signaling molecules such as nuclear factor-kappa B(NF-120581B) [25 26]

In our study we found that VIP downregulated theNKG2D DAP10 and NF-120581B expression in NK cells whichcould be reversed completely or mostly by VIP antagonist


Table6Th

eexpressionof

NKG

2DD

AP10andNF-120581Bin

NKcells

incubatedwith

drug

Group

nNKG

2Dproteinexpressio

nNKG

2DmRN

ADAP10proteinexpressio

nDAP10mRN

ANF-120581Bproteinexpressio

nNF-120581Bp6

5mRN

Aminus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)

A10

07

30

0232plusmn0024

00

82

0415plusmn0009

17

20

0201plusmn0014

B10

01

81

0554plusmn0031

00

37

0617plusmn0013

00

82

0495plusmn0015

C10

00

46

0742plusmn0080

00

28

0625plusmn0022

00

55

0504plusmn0027

119867=16447

119865=227967

119867=8136

119865=514408

119867=18051

119865=680369

119875=0000

119875=0000

119875=0017

119875=0000

119875=0000

119875=0000

Group

AN

Kcells

+VIPgroup

BNKcells

+VIP

+VIP

antagonist

andgrou

pC

NKcells

(con

trol)

IODthe

integrated

opticaldensity

ratio

ofthetargetgenes

(NKG

2DD

AP10NF-120581B)

to120573-Actin


(a)

(b)

(c)

Figure 4 NKG2D protein expression in different groups times200 ((a)NK + VIP (b) NK + VIP + VIP antagonist and (c) NK)

Moreover we found that the alteration trend of the expres-sions of NKG2D DAP10 and NF-120581B p65 was similar soNKG2DDAP10NF-120581B might be a pathway by which VIPaffected NK cells NF-120581B was a transcription factor whichwas heterodimers or homodimers consisted of the membersof the NF-120581B family It was a critical regulator of multiplebiological functions such as affecting innate and adaptiveimmunity affecting cell survival So NF-120581BNKG2DDAP10might be another pathway by which VIP affected NK cellsThey need further confirmation

In our study we need to attain highly purified NKcells It has been reported that NK cells could be separatedby immunomagnetic beads sorting system [27] and flowcytometer (FCM) [28] However those methods mentionedabove were expensive and complicated In our study weused complement lysis (CDC) method to separate NK cellsWe found that the NK cells purity raised from 18 (beforepurification) to 60 (purified) This method was reported byCiccone E [19] He found that the purity surviva rate andcytotoxic activity of NK cells were good when using CDCmethod There were TB lymphocytes in the NK solution

(a)

(b)

(c)

Figure 5 DAP10 protein expression in different groups times200 ((a)NK + VIP (b) NK + VIP + VIP antagonist and (c) NK)

except NK cells but these cells might did not affect experi-ment results for follow reason It was reported that NKG2Dwas also expressed on most NKT cells macrophage cellsand subpopulations of 120574120575T cells except NK cells NKT cellsmacrophage cells and 120574120575T cells could also kill tumor cellslike NK cells [23 29ndash31] but the concentration of NKT and120574120575T cells in PBMC is rare Macrophage cells were adherentgrowth We gained the NK cells solution from supernatantand liquor Thus these cells might not affect experimentalresults also We also found that the concentration of NK cells(18)was a little higher than normal range (5ndash10) in PBMCwhich might be the effect of rhIL-2 and PHA added intoPBMC solution

There are three types of VIPPACAP receptors VPAC1VPAC2 and PAC1 The different receptors bond VIP withdifferent affinities VPAC1 showed the most remarkableaffinities to VIP among all the receptors [11 21] So VPAC1might play amajor role In this study we observed the VPAC1in NK andMKN45 cells However if any other VIP receptorstook role in this signal pathway they would need furtherstudy


(a)

(b)

(c)

Figure 6 NF-120581B protein expression in different groups times200 ((a)NK + VIP (b) NK + VIP + VIP antagonist and (c) NK)

From above we concluded that the VIP could inhibitNK cellsrsquo cytotoxicity on MKN45 which might get throughdownregulating the expressions of NKG2D DAP10 and NF-120581B in NK cells So gastric cancer cells might escape organismimmune cleaning by secreting VIP to inhibit NKG2D signalpathway of NK cells However are there any other membersexcept NKG2D DAP10 and NF-120581B in this signal pathwayHow did NK cells affect gastric adenocarcinoma in vivoThose questions need further study Our finding provide anew insight into the importance of VIP in gastric cancerprogression The VIP antagonist may be helpful antigastriccancer drug by targeted VIP

Conflict of Interests

The authors declare that there is no conflict of interests inwriting this paper

Authorsrsquo Contribution

Chong Wang and Xi-Jin Zhou contributed equally to thisstudy and share first authorship

M C A B

600

500

400

300

200

(a)

M C A B

(b)

M C A B

(c)

Figure 7 Electrophoresis of PCR product of NKG2D DAP10 andNF-120581B mRNA in different groups (a) stands for NKG2D (b) forDAP10 and (c) for NF-120581B M stands for marker DL600 C for NKcells A forNKcells +VIP andB forNKcells +VIP+VIP antagonistThe length of PCR products of NKG2D DAP10 and NF-120581B was365 bp 282 bp and 326 bp

Acknowledgments

This study was supported by the Nation Nature ScienceFund of China (Grant no 30760086) and the First AffiliatedHospital of Nanchang University

References

[1] M A Caligiuri ldquoHuman natural killer cellsrdquo Blood vol 112 no3 pp 461ndash469 2008

[2] A M Jamieson A Diefenbach C W McMahon N XiongJ R Carlyle and D H Raulet ldquoThe role of the NKG2Dimmunoreceptor in immune cell activation and natural killingrdquoImmunity vol 17 no 1 pp 19ndash29 2002

[3] N Guerra Y X Tan N T Joncker et al ldquoNKG2D-deficientmice are defective in tumor surveillance in models of sponta-neous malignancyrdquo Immunity vol 28 no 4 pp 571ndash580 2008

[4] D D Billadeau J L Upshaw R A Schoon C J Dickand P J Leibson ldquoNKG2D-DAP10 triggers human NK cell-mediated killing via a Syk-independent regulatory pathwayrdquoNature Immunology vol 4 no 6 pp 557ndash564 2003

[5] D Garrity M E Call J Feng and K W Wucherpfennig ldquoTheactivating NKG2D receptor assembles in the membrane withtwo signaling dimers into a hexameric structurerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 102 no 21 pp 7641ndash7646 2005


[6] Y Fukumoto M Ikeguchi S Matsumoto et al ldquoDetection ofcancer cells and gene expression of cytokines in the peritonealcavity in patients with gastric cancerrdquoGastric Cancer vol 9 no4 pp 271ndash276 2006

[7] S I Said and V Mutt ldquoPolypeptide with broad biologicalactivity isolation from small intestinerdquo Science vol 169 no3951 pp 1217ndash1218 1970

[8] M Delgado and D Ganea ldquoVasoactive intestinal peptidea neuropeptide with pleiotropic immune functionsrdquo AminoAcids vol 45 no 1 pp 25ndash39 2013

[9] A Valdehita M J Carmena A M Bajo and J C Prieto ldquoRNAinterference-directed silencing of VPAC1 receptor inhibits VIPeffects on both EGFR and HER2 transactivation and VEGFsecretion in human breast cancer cellsrdquoMolecular and CellularEndocrinology vol 348 no 1 pp 241ndash246 2012

[10] G H Li W Qian G Q Song and X H Hou ldquoEffectof vasoactive intestinal peptide on gastric adenocarcinomardquoJournal of Gastroenterology and Hepatology vol 22 no 8 pp1328ndash1335 2007

[11] I Langer and P Robberecht ldquoMolecular mechanisms involvedin vasoactive intestinal peptide receptor activation and regula-tion current knowledge similarities to and differences from theA family of G-protein-coupled receptorsrdquo Biochemical SocietyTransactions vol 35 part 4 pp 724ndash728 2007

[12] S Schulz C Rocken C Mawrin W Weise V Hollt andS Schulz ldquoImmunocytochemical identification of VPAC1VPAC2 and PAC1 receptors in normal and neoplastic humantissues with subtype-specific antibodiesrdquo Clinical CancerResearch vol 10 no 24 pp 8235ndash8242 2004

[13] S S Diebold ldquoActivation of dendritic cells by toll-like receptorsand C-type lectinsrdquo in Handbook of Experimental Pharmacol-ogy vol 188 pp 3ndash30 2009

[14] A Chorny E Gonzalez-Rey A Fernandez-Martin D PozoD Ganea and M Delgado ldquoVasoactive intestinal peptideinduces regulatory dendritic cells with therapeutic effects onautoimmune disordersrdquo Proceedings of the National Academyof Sciences of the United States of America vol 102 no 38 pp13562ndash13567 2005

[15] E J Goetzl R C Chan and M Yadav ldquoDiverse mechanismsand consequences of immunoadoption of neuromediator sys-temsrdquoAnnals of the New York Academy of Sciences vol 1144 pp56ndash60 2008

[16] B Lv Y Tang F Chen and X Xiao ldquoVasoactive intestinalpeptide and pituary adenylate cyclase-activating polypeptideinhibit tissue factor expression in monocyte in vitro and invivordquo Shock vol 31 no 2 pp 185ndash191 2009

[17] EA Szliter S Lighvani R P Barrett andLDHazlett ldquoVasoac-tive intestinal peptide balances pro- and anti-inflammatorycytokines in the Pseudomonas aeruginosa-infected cornea andprotects against corneal perforationrdquo Journal of Immunologyvol 178 no 2 pp 1105ndash1114 2007

[18] M Hejna G Hamilton T Brodowicz et al ldquoSerum levels ofvasoactive intestinal peptide (VIP) in patients with adenocarci-nomas of the gastrointestinal tractrdquoAnticancer Research vol 21no 2 pp 1183ndash1188 2001

[19] E Ciccone D Pende O Viale et al ldquoSpecific recognition ofhuman CD3minusCD16+ natural killer cells requires the expressionof an autosomic recessive gene on target cellsrdquo Journal ofExperimental Medicine vol 172 no 1 pp 47ndash52 1990

[20] M A Tzaneva ldquoEndocrine cells in gastric carcinoma andadjacentmucosa An immunohistochemical and ultrastructuralstudyrdquoHistochemical Journal vol 34 no 3-4 pp 173ndash180 2002

[21] M Yadav and E J Goetzl ldquoVasoactive intestinal peptide-mediated Th17 differentiation an expanding spectrum ofvasoactive intestinal peptide effects in immunity and autoim-munityrdquo Annals of the New York Academy of Sciences vol 1144pp 83ndash89 2008

[22] M Champsaur and L L Lanier ldquoEffect of NKG2D ligandexpression on host immune responsesrdquo Immunological Reviewsvol 235 no 1 pp 267ndash285 2010

[23] D H Raulet ldquoRoles of the NKG2D immunoreceptor and itsligandsrdquoNature Reviews Immunology vol 3 no 10 pp 781ndash7902003

[24] B Zafirova FMWensveenMGulin and B Polic ldquoRegulationof immune cell function and differentiation by the NKG2Dreceptorrdquo Cellular and Molecular Life Sciences vol 68 no 21pp 3519ndash3529 2011

[25] C M Tato N Mason D Artis et al ldquoOpposing roles of NF-120581Bfamily members in the regulation of NK cell proliferation andproduction of IFN-120574rdquo International Immunology vol 18 no 4pp 505ndash513 2006

[26] F Wan and M J Lenardo ldquoThe nuclear signaling of NF-120581Bcurrent knowledge new insights and future perspectivesrdquo CellResearch vol 20 no 1 pp 24ndash33 2010

[27] Y Nakashima M Deie S Yanada P Sharman and M OchildquoMagnetically labeled human natural killer cells accumulatedin vitro by an external magnetic force are effective against HOSosteosarcoma cellsrdquo International Journal of Oncology vol 27no 4 pp 965ndash971 2005

[28] A Piroozmand and Z M Hassan ldquoEvaluation of natural killercell activity in pre and post treated breast cancer patientsrdquoJournal of Cancer Research and Therapeutics vol 6 no 4 pp478ndash481 2010

[29] Y Yoshida J Nakajima H Wada and K Kakimi ldquo120574120575 T-cellimmunotherapy for lung cancerrdquo Surgery Today vol 41 no 5pp 606ndash611 2011

[30] K Maasho J Opoku-Anane A I Marusina J E Coligan andF Borrego ldquoCutting edge NKG2D is a costimulatory receptorfor human naive CD8+ T cellsrdquo Journal of Immunology vol 174no 8 pp 4480ndash4484 2005

[31] M Girardi D E Oppenheim C R Steele et al ldquoRegulation ofcutaneous malignancy by 120574120575 T cellsrdquo Science vol 294 no 5542pp 605ndash609 2001

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


NK cell activity So VIP is the most important immuneinhibiting neuropeptide Many studies showed that VIP wasrelated with the gastric cancer Hejna et al reported that theserum VIP concentration increased in patients with gastriccancer [18]We have showed that the gastric adenocarcinomatissues contained secreting VIP cancer cells [10] Above thissuggested that VIPmight play an important role in inhibitingorganism immune functions

So we suppose that VIP which is secreted by gastriccancer cells may facilitate gastric cancer cells to escapeorganism immune cleaning by inhibiting NKG2D signalmolecules in NK cells To confirm it we want to research theinfluence of VIP on NK cells cytotoxicity to kill the gastricadenocarcinoma cell line (MKN45 cells) in vitro and researchthe relationship of this influence with the NKG2D signalmolecules in NK cells

2 Materials and Methods

21 Natural Killer (NK) Cells Separation Purification andIdentification Heparinized bloodwas collected between 600and 1000 am from 10 healthy volunteers of the staff at theNanchang University (mean age 426 plusmn 84 60 male nohistory of chronic illness and without current prescriptionmedications usage) The peripheral blood mononuclear cells(PBMC) were obtained by standard Ficoll-Hypaque den-sity gradient centrifugation and suspended by RPMI-1640media with 10 heat-inactivated fetal bovine serum (FBS)(Gibco Invitrogen Grand Island NY USA) The activatedNK cells were purified from PBMC by complement lysis(CDC) method [19] and cultured in RPMI-1640 mediumwith 10 newborn calf serum (NBCS) (Gibco InvitrogenGrand Island NY USA) 1 times 106UL recombinant humaninterleukin-2 (rhIL-2 PeproTech Rocky Hill NJ USA) and10mgL phytohaemagglutinin (PHA Sigma St Louis MOUSA) under a microaerophilic environment (at 37∘C 2O2 5 CO

2) The NK cells (expressed CD3minusCD16+CD56+)

percentage was detected by FACS after incubated with anti-human CD3 FITC(CD16+CD56) PE Cocktail (BioLegendSan Diego CA USA) according to its instruction

22 Cell Culture The MKN45 cell line originated fromhuman poorly differentiated gastric adenocarcinoma (fromATCC company) The cells were cultured in RPMI-1640medium with 10 NBCS under a microaerophilic environ-ment (at 37∘C 2 O

2 5 CO

2)

23 The Cytotoxicity of NK Cells on the Growth of MKN45Cells The MKN45 cells were cultured in the medium ina 96-well plate Each well contained 200 120583L cancer cellssolution in a 1 times 104 cellsmL concentration The supernatewas thrown away after the MKN45 cells were cultured for4 h then each well was added NK cells andor different drugsolutions for different incubated time according to differentgroups as follows Each plate included 4 wells for the negativecontrols (seeded byMKN45 andmedium) and 4 wells for theblank controls (only seeded by 200 120583L medium) Each group

included 4 wells at least in each time Each test repeated threetimes

231 Effect of VIP on the Growth of NK Cells to MKN45 CellsIn this test the MKN45 cells in 96-well plate were added180 120583L NK cells solution in a 1 times 105 cellsmL concentration(the ratio of effect to target was 10 1) and 20120583L VIP solutionsto each well for 48 h The final concentration of VIP in eachwell was 1 times 10minus5molL to 1 times 10minus7molL respectively

232 Interaction between VIP and Its Antagonist In thistest the MKN45 cells in 96-well plate were added 180 120583LNK cells solution in a 1 times 105 cellsmL concentration and20120583L different drug solutions to each well for 48 h The drugsolution included VIP its antagonist ([D-p-Cl-Phe6 Leu17]-VIP Sigma St Louis MO USA) or VIP combined with itsantagonist The final concentration of VIP in each well was 1times 10minus6molL its antagonist in each well was 1 times 10minus4 1 times 10minus5or 1 times 10minus6molL respectively

233 MTT The growth state of MKN45 cells in eachtest mentioned above were measured by methyl thiazolyldiphenyl tetrazolium bromide assay (MTT) The OD value(OD490

) in MTT represented the quantity of living MKN45cells but not NK cells because the suspending NK cells werethrown away in the course of removing supernate duringMTT test

24 Effect of VIP or Its Antagonist on NKG2D SignalMoleculesof NK Cells NK cells were cultured in the medium in a 6-well plate Each well contained 2970 120583L NK cells solutionin a 1 times 106 cellsmL and 30120583L different drug solutions toeach well The drug solution included VIP its antagonistor mixed solution of VIP and its antagonist The finalconcentration of VIP and its antagonist in each well was10minus6molL and 10minus5molL respectively After incubation for48 h the NK cells were used to detect target gene mRNA andprotein expressions by RT-PCR or immunocytochemistrytest Each group included 3 wells at least in each test (negativecontrol group contained 2970 120583L NK cells solution in a 1times 106 cellsmL concentration and 30120583L medium) The testrepeated three times

241 RNA Isolation and Reverse Transcription PolymeraseChain Reaction (RT-PCR)

RNA Isolation After NK cells were incubated by the VIP andits antagonist for 48 h as mentioned above the total RNA ofthe NK cells was extracted according to the procedure of theTrizol reagent (TianGen Biotechnology Co Beijing China)

RT-PCR A typical 25-120583L reaction contained 10120583L totalRNA 200 units of Moloney murine leukemia virus (MMLV)reverse transcriptase 500 ng oligo(dT)

15 1 times 5 120583L reverse

transcription buffer 10mmolL deoxy-ribonucleoside tri-phosphate (dNTP) RNasin inhibitor (Promega Madison























3 Results





490value was












100

101

102

103

104

100

101

102

103

104

FL1-height

FL2

-hei

ght

Quad Gated ()

UL

UR

LL

LR

018

012

9923

048

(a)

100

101

102

103

104

100

101

102

103

104

NK

CD3 FITC

6005

2385

890

720

Quad

UL

UR

LL

LR

Gated ()

(b)

100

101

102

103

104

100

101

102

103

104

NK

CD3 FITC

1527

1672

3498

3303

Quad

UL

UR

LL

LR

Gated ()

(c)





2= 36750

B 10 18508 119875 lt 001



Group n OD490







G MKN45 + NK 12 0106 plusmn 0016a




Group n OD490


acd


acd


abce






I MKN45 + NK 12 0104 plusmn 0024a






490values


490






4 Discussion





Time (h) n OD490




0200 plusmn 0028

0106 plusmn 0016






(a)

(b)






M K C

600

500

400

300

200

(a)

M K C

(b)

M K C B A

(c)





Table6Th

eexpressionof

NKG

2DD

AP10andNF-120581Bin

NKcells

incubatedwith

drug

Group

nNKG

2Dproteinexpressio

nNKG

2DmRN


nDAP10mRN


nNF-120581Bp6

5mRN

Aminus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)

A10

07

30

0232plusmn0024

00

82

0415plusmn0009

17

20

0201plusmn0014

B10

01

81

0554plusmn0031

00

37

0617plusmn0013

00

82

0495plusmn0015

C10

00

46

0742plusmn0080

00

28

0625plusmn0022

00

55

0504plusmn0027

119867=16447

119865=227967

119867=8136

119865=514408

119867=18051

119865=680369

119875=0000

119875=0000

119875=0017

119875=0000

119875=0000

119875=0000

Group

AN

Kcells

+VIPgroup

BNKcells

+VIP

+VIP

antagonist

andgrou

pC

NKcells

(con

trol)

IODthe

integrated

opticaldensity

ratio

ofthetargetgenes

(NKG

2DD

AP10NF-120581B)

to120573-Actin


(a)

(b)

(c)




(a)

(b)

(c)





(a)

(b)

(c)







M C A B

600

500

400

300

200

(a)

M C A B

(b)

M C A B

(c)


Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































3 Results





490value was












100

101

102

103

104

100

101

102

103

104

FL1-height

FL2

-hei

ght

Quad Gated ()

UL

UR

LL

LR

018

012

9923

048

(a)

100

101

102

103

104

100

101

102

103

104

NK

CD3 FITC

6005

2385

890

720

Quad

UL

UR

LL

LR

Gated ()

(b)

100

101

102

103

104

100

101

102

103

104

NK

CD3 FITC

1527

1672

3498

3303

Quad

UL

UR

LL

LR

Gated ()

(c)





2= 36750

B 10 18508 119875 lt 001



Group n OD490







G MKN45 + NK 12 0106 plusmn 0016a




Group n OD490


acd


acd


abce






I MKN45 + NK 12 0104 plusmn 0024a






490values


490






4 Discussion





Time (h) n OD490




0200 plusmn 0028

0106 plusmn 0016






(a)

(b)






M K C

600

500

400

300

200

(a)

M K C

(b)

M K C B A

(c)





Table6Th

eexpressionof

NKG

2DD

AP10andNF-120581Bin

NKcells

incubatedwith

drug

Group

nNKG

2Dproteinexpressio

nNKG

2DmRN


nDAP10mRN


nNF-120581Bp6

5mRN

Aminus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)

A10

07

30

0232plusmn0024

00

82

0415plusmn0009

17

20

0201plusmn0014

B10

01

81

0554plusmn0031

00

37

0617plusmn0013

00

82

0495plusmn0015

C10

00

46

0742plusmn0080

00

28

0625plusmn0022

00

55

0504plusmn0027

119867=16447

119865=227967

119867=8136

119865=514408

119867=18051

119865=680369

119875=0000

119875=0000

119875=0017

119875=0000

119875=0000

119875=0000

Group

AN

Kcells

+VIPgroup

BNKcells

+VIP

+VIP

antagonist

andgrou

pC

NKcells

(con

trol)

IODthe

integrated

opticaldensity

ratio

ofthetargetgenes

(NKG

2DD

AP10NF-120581B)

to120573-Actin


(a)

(b)

(c)




(a)

(b)

(c)





(a)

(b)

(c)







M C A B

600

500

400

300

200

(a)

M C A B

(b)

M C A B

(c)


Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















100

101

102

103

104

100

101

102

103

104

FL1-height

FL2

-hei

ght

Quad Gated ()

UL

UR

LL

LR

018

012

9923

048

(a)

100

101

102

103

104

100

101

102

103

104

NK

CD3 FITC

6005

2385

890

720

Quad

UL

UR

LL

LR

Gated ()

(b)

100

101

102

103

104

100

101

102

103

104

NK

CD3 FITC

1527

1672

3498

3303

Quad

UL

UR

LL

LR

Gated ()

(c)





2= 36750

B 10 18508 119875 lt 001



Group n OD490







G MKN45 + NK 12 0106 plusmn 0016a




Group n OD490


acd


acd


abce






I MKN45 + NK 12 0104 plusmn 0024a






490values


490






4 Discussion





Time (h) n OD490




0200 plusmn 0028

0106 plusmn 0016






(a)

(b)






M K C

600

500

400

300

200

(a)

M K C

(b)

M K C B A

(c)





Table6Th

eexpressionof

NKG

2DD

AP10andNF-120581Bin

NKcells

incubatedwith

drug

Group

nNKG

2Dproteinexpressio

nNKG

2DmRN


nDAP10mRN


nNF-120581Bp6

5mRN

Aminus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)

A10

07

30

0232plusmn0024

00

82

0415plusmn0009

17

20

0201plusmn0014

B10

01

81

0554plusmn0031

00

37

0617plusmn0013

00

82

0495plusmn0015

C10

00

46

0742plusmn0080

00

28

0625plusmn0022

00

55

0504plusmn0027

119867=16447

119865=227967

119867=8136

119865=514408

119867=18051

119865=680369

119875=0000

119875=0000

119875=0017

119875=0000

119875=0000

119875=0000

Group

AN

Kcells

+VIPgroup

BNKcells

+VIP

+VIP

antagonist

andgrou

pC

NKcells

(con

trol)

IODthe

integrated

opticaldensity

ratio

ofthetargetgenes

(NKG

2DD

AP10NF-120581B)

to120573-Actin


(a)

(b)

(c)




(a)

(b)

(c)





(a)

(b)

(c)







M C A B

600

500

400

300

200

(a)

M C A B

(b)

M C A B

(c)


Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















2= 36750

B 10 18508 119875 lt 001



Group n OD490







G MKN45 + NK 12 0106 plusmn 0016a




Group n OD490


acd


acd


abce






I MKN45 + NK 12 0104 plusmn 0024a






490values


490






4 Discussion





Time (h) n OD490




0200 plusmn 0028

0106 plusmn 0016






(a)

(b)






M K C

600

500

400

300

200

(a)

M K C

(b)

M K C B A

(c)





Table6Th

eexpressionof

NKG

2DD

AP10andNF-120581Bin

NKcells

incubatedwith

drug

Group

nNKG

2Dproteinexpressio

nNKG

2DmRN


nDAP10mRN


nNF-120581Bp6

5mRN

Aminus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)

A10

07

30

0232plusmn0024

00

82

0415plusmn0009

17

20

0201plusmn0014

B10

01

81

0554plusmn0031

00

37

0617plusmn0013

00

82

0495plusmn0015

C10

00

46

0742plusmn0080

00

28

0625plusmn0022

00

55

0504plusmn0027

119867=16447

119865=227967

119867=8136

119865=514408

119867=18051

119865=680369

119875=0000

119875=0000

119875=0017

119875=0000

119875=0000

119875=0000

Group

AN

Kcells

+VIPgroup

BNKcells

+VIP

+VIP

antagonist

andgrou

pC

NKcells

(con

trol)

IODthe

integrated

opticaldensity

ratio

ofthetargetgenes

(NKG

2DD

AP10NF-120581B)

to120573-Actin


(a)

(b)

(c)




(a)

(b)

(c)





(a)

(b)

(c)







M C A B

600

500

400

300

200

(a)

M C A B

(b)

M C A B

(c)


Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Time (h) n OD490




0200 plusmn 0028

0106 plusmn 0016






(a)

(b)






M K C

600

500

400

300

200

(a)

M K C

(b)

M K C B A

(c)





Table6Th

eexpressionof

NKG

2DD

AP10andNF-120581Bin

NKcells

incubatedwith

drug

Group

nNKG

2Dproteinexpressio

nNKG

2DmRN


nDAP10mRN


nNF-120581Bp6

5mRN

Aminus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)

A10

07

30

0232plusmn0024

00

82

0415plusmn0009

17

20

0201plusmn0014

B10

01

81

0554plusmn0031

00

37

0617plusmn0013

00

82

0495plusmn0015

C10

00

46

0742plusmn0080

00

28

0625plusmn0022

00

55

0504plusmn0027

119867=16447

119865=227967

119867=8136

119865=514408

119867=18051

119865=680369

119875=0000

119875=0000

119875=0017

119875=0000

119875=0000

119875=0000

Group

AN

Kcells

+VIPgroup

BNKcells

+VIP

+VIP

antagonist

andgrou

pC

NKcells

(con

trol)

IODthe

integrated

opticaldensity

ratio

ofthetargetgenes

(NKG

2DD

AP10NF-120581B)

to120573-Actin


(a)

(b)

(c)




(a)

(b)

(c)





(a)

(b)

(c)







M C A B

600

500

400

300

200

(a)

M C A B

(b)

M C A B

(c)


Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Table6Th

eexpressionof

NKG

2DD

AP10andNF-120581Bin

NKcells

incubatedwith

drug

Group

nNKG

2Dproteinexpressio

nNKG

2DmRN


nDAP10mRN


nNF-120581Bp6

5mRN

Aminus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)minus

+++

+++

expressio

n(IOD)

A10

07

30

0232plusmn0024

00

82

0415plusmn0009

17

20

0201plusmn0014

B10

01

81

0554plusmn0031

00

37

0617plusmn0013

00

82

0495plusmn0015

C10

00

46

0742plusmn0080

00

28

0625plusmn0022

00

55

0504plusmn0027

119867=16447

119865=227967

119867=8136

119865=514408

119867=18051

119865=680369

119875=0000

119875=0000

119875=0017

119875=0000

119875=0000

119875=0000

Group

AN

Kcells

+VIPgroup

BNKcells

+VIP

+VIP

antagonist

andgrou

pC

NKcells

(con

trol)

IODthe

integrated

opticaldensity

ratio

ofthetargetgenes

(NKG

2DD

AP10NF-120581B)

to120573-Actin


(a)

(b)

(c)




(a)

(b)

(c)





(a)

(b)

(c)







M C A B

600

500

400

300

200

(a)

M C A B

(b)

M C A B

(c)


Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

(b)

(c)




(a)

(b)

(c)





(a)

(b)

(c)







M C A B

600

500

400

300

200

(a)

M C A B

(b)

M C A B

(c)


Acknowledgments


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

(b)

(c)







M C A B

600

500

400

300

200

(a)

M C A B

(b)

M C A B

(c)


Acknowledgments


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 Effect of Vasoactive Intestinal Peptide (VIP) on … · 2019. 7. 31. · er NK cells were incubated with the VIP, its antagonist for h in -well plate as mentioned