Molecular Cancer BioMed Central - COnnecting REpositories · data set so as to identify differentially expressed colon transcripts in rats fed SPI, WPH or CAS; results are reported

BioMed CentralMolecular Cancer

Open AcceResearchDietary exposure to soy or whey proteins alters colonic global gene expression profiles during rat colon tumorigenesisRijin Xiao12 Thomas M Badger12 and Frank A Simmen12

Address 1Arkansas Childrens Nutrition Center 1120 Marshall Street Little Rock AR 72202 USA and 2Department of Physiology and Biophysics University of Arkansas for Medical Sciences Little Rock AR 72202 USA

Email Rijin Xiao - XiaoRijinuamsedu Thomas M Badger - BadgerThomasMuamsedu Frank A Simmen - SimmenFrankAuamsedu

Corresponding author

colon cancersoywheygene expression profilingneuro-endocrinemicroarrayrat

AbstractBackground We previously reported that lifetime consumption of soy proteins or whey proteinsreduced the incidence of azoxymethane (AOM)-induced colon tumors in rats To obtain insightsinto these effects global gene expression profiles of colons from rats with lifetime ingestion ofcasein (CAS control diet) soy protein isolate (SPI) and whey protein hydrolysate (WPH) dietswere determined

Results Male Sprague Dawley rats fed one of the three purified diets were studied at 40 weeksafter AOM injection and when tumors had developed in some animals of each group Total RNApurified from non-tumor tissue within the proximal half of each colon was used to preparebiotinylated probes which were hybridized to Affymetrix RG_U34A rat microarrays containingprobes sets for 8799 rat genes Microarray data were analyzed using DMT (Affymetrix) SAM(Stanford) and pair-wise comparisons Differentially expressed genes (SPI andor WPH vs CAS)were found We identified 31 induced and 49 repressed genes in the proximal colons of the SPI-fed group and 44 induced and 119 repressed genes in the proximal colons of the WPH-fed grouprelative to CAS Hierarchical clustering identified the co-induction or co-repression of multiplegenes by SPI and WPH The differential expression of I-FABP (292- 397-fold down-regulated inSPI and WPH fed rats P = 0023 P = 001 respectively) cyclin D1 (161- 242-fold down-regulatedin SPI and WPH fed rats P = 0033 P = 0001 respectively) and the c-neu proto-oncogene (246- 410-fold down-regulated in SPI and WPH fed rats P lt 0001 P lt 0001 respectively) mRNAswere confirmed by real-time quantitative RT-PCR SPI and WPH affected colonic neuro-endocrinegene expression peptide YY (PYY) and glucagon mRNAs were down-regulated in WPH fed ratswhereas somatostatin mRNA and corresponding circulating protein levels were enhanced by SPIand WPH

Conclusions The identification of transcripts co- or differentially-regulated by SPI and WPH dietssuggests common as well as unique anti-tumorigenesis mechanisms of action which may involvegrowth factor neuroendocrine and immune system genes SPI and WPH induction of somatostatina known anti-proliferative agent for colon cancer cells would inhibit tumorigenesis

Published 11 January 2005

Molecular Cancer 2005 41 doi1011861476-4598-4-1

Received 08 September 2004Accepted 11 January 2005

This article is available from httpwwwmolecular-cancercomcontent411

copy 2005 Xiao et al licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (httpcreativecommonsorglicensesby20) which permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

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Molecular Cancer 2005 41 httpwwwmolecular-cancercomcontent411

BackgroundColorectal cancer (CRC) is the third most common cancerand the third leading cause of cancer-related mortality inthe US [12] Estimated new cases of colon cancer were79650 for men and 73530 for women in 2004 [1]approximately $63 billion is spent in the United Stateseach year on treatment of CRC [2] Accumulating evi-dence suggests that diet is an important environmentalfactor in the etiology of CRC High consumption of redmeats animal fats chocolate alcohol and refined cerealsare linked to higher incidence of these cancers in Westernsocieties [3-5] whereas protective effects of fruits vegeta-bles and whole grains have been suggested [5]

Soy foods and soybean constituents have received consid-erable attention for their potential role in reducing cancerrisk [67] Our laboratories reported the protective effectsof lifetime ingestion of soy protein isolate (SPI) onazoxymethane (AOM)-induced colon cancer in rats [8]Similarly the effect of whey protein hydrolysate (WPH) inthe diet to reduce colon tumor incidence has beenreported by us and others [9-11] Several hypotheses havebeen proposed to account for soy and whey protein-induced anti-tumorigenesis For example soy isoflavoneshave been proposed to play a key role in soys anti-cancerfunctions [12] Yanagihara et al among others reportedthat genistein inhibits colon cancer cell proliferation andstimulates apoptosis in vitro [13-15] However subcuta-neous administration of genistein to mice did not confirmthese in vitro effects [16] Holly et al reported that soysphingolipids inhibit colonic cell proliferation and sug-gested that this may partially account for its anticancerbenefits [17] Other reports indicate that soy diets inhibittumorigenesis by regulating the synthesis or activities ofspecific proteins For example Rowlands et al reportedthat dietary soy and whey proteins down-regulate expres-sion of liver and mammary gland phase I enzymesinvolved in carcinogen activation [18] Elevated activitiesof phase II detoxification enzymes were reported in soy-fed rats [1920] Such dietary effects may result in lowertissue concentrations of activated carcinogen The antican-cer properties of whey proteins have been ascribed to theirability to elevate cellular levels of the antioxidant glutath-ione [2122] Moreover the whey protein α-lactalbumininhibits proliferation of mammary epithelial cells in vitro[11] The anticancer properties of whey may also relate toits immune system-enhancing actions [23]

Despite extensive research there is no consensus for anti-cancer mechanism(s) of soy and whey which willundoubtedly involve multiple interrelated processespathways and many components Many of the samemolecular and biochemical changes underlying humancolon cancer are observed in the azoxymethane (AOM)-induced rat colon cancer model [24] Moreover previous

studies suggest a different molecular etiology for tumorsof the proximal and distal colon in this model and inhuman colon [2425] Differential dietary effects on prox-imal vs distal colon DNA damage were noted [26] andWesternization of the human diet is thought to havefavored a shift of tumors from distal to more proximallocations [27] Thus region-specific localization of dietaryeffects on colon tumorigenesis is an important factor toconsider in any molecular analysis of CRC Here we useAffymetrix high-density oligonucleotide microarrays todetermine the expression profiles of non-tumor (ie nor-mal) tissue in proximal colons (PC) of rats subjected tolifetime diets containing casein (CAS control diet) soyprotein isolate (SPI) or whey protein hydrolysate (WPH)and which were administered AOM to induce tumors Wehypothesized that genes whose expression contributes toanti-tumorigenesis would be regulated in parallel by SPIand WPH in addition changes unique to each diet mightalso be apparent

ResultsValidation of the microarray approachQuality control steps ensured that the RNA used formicroarray and real-time RT-PCR analysis was of highquality These steps included evaluation of the RNA withthe RNA 6000 Nano Assay and assessment of the cRNAhybridization to GeneChips by comparison of dataobtained for probe sets representative of 5 and 3 ends ofcontrol genes All RNA samples had an A260280 absorb-ance ratio between 19 and 21 The ratio of 28S to 18SrRNA was very close to 2 on RNA electropherograms andsignal ratios below 3 were noted for 3 vs 5 probe sets forβ-actin and glyceraldehyde-3-phosphate dehydrogenase(per Affymetrix user guidelines) after hybridization

Total false change rates (TFC) were determined followingAffymetrix-recommended guidelines [28] except that theinter-chip comparisons used cRNA targets made in paral-lel starting from the same RNA pool Inter-chip variabilitymeasured as TFC was 025 ndash 06 and well below thesuggested 2 cutoff (Table 1) These values confirmed thefidelity and reproducibility of the microarray procedures

Table 1 Inter-chip variability

Diet group Number of arrays TFC ()

CAS 3 0252 plusmn 0138WPH 3 0369 plusmn 0025SPI 3 0570 plusmn 0165

TFC (Total false change) = false change rate (decreased category) + false change rate (increased category) as described in ref 28 TFC reported as mean plusmn SEM TFC should be no more than 2 (Affymetrix)

used Unsupervised nearest-neighbor hierarchical cluster-ing identified differences in proximal colon gene expres-sion profiles of CAS SPI and WPH groups (Figs 1 and 2)indicating that the type of dietary protein has a major

effect on gene expression in normal proximal colon tissueof AOM-treated rats Interestingly the overall gene expres-sion profiles for SPI and WPH groups were more similarto each other than each was to the CAS group (Fig 1A)

Differentially expressed genesMultiple filtering criteria were applied to the microarraydata set so as to identify differentially expressed colontranscripts in rats fed SPI WPH or CAS results arereported only for transcripts that passed all three analyti-cal filters used DMT t-test SAM and pair-wise comparisonsurvival methods Among the 8799 genes and ESTs exam-ined with the rat U34A array we identified 31 inducedand 49 repressed genes in proximal colons of SPI-fed ratswhereas 44 induced and 119 repressed genes weredetected in WPH-fed rats (Tables 2 3 4 5) Interestingly

Hierarchical clustering of proximal colon gene expression profilesFigure 1Hierarchical clustering of proximal colon gene expression profiles A Clustering of nine PC global gene expression profiles (8799 genes) n = 3 profiles each for CAS SPI and WPH Each cell represents the expression level of an individ-ual gene in each sample (green = low expression black = middle expression red = high expression) The dendrogram reflects the extent of relatedness of different profiles the shorter branch-point of the SPI and WPH trees indicates the greater similarity between these profiles B Clustering of 18 global comparative expression profiles including 9 of SPI vs CAS and 9 of WPH vs CAS profiles Each row in the heat map represents the relative expression level of a given gene across all comparisons (red = up regulated black = unchanged green = down regulated)

A Hierarchical clustering of expression profiles

CAS1 CAS2 CAS3 SPI3 SPI1 SPI2 WPH1 WPH3 WPH2

B Hierarchical clustering of comparative analysis profiles

Hierarchical clustering of 211 differentially expressed genes in either SPI or WPHFigure 2Hierarchical clustering of 211 differentially expressed genes in either SPI or WPH The differential expression data are taken only from the pairwise comparison analysis with CAS profiles used as baseline Each cell in the heat map represents the relative expression level of a given gene in an individual comparison analysis (red = up regulated black = unchanged green = down regulated) The dendrogram reflects the relat-edness of different profiles

Hierarchical clustering of regulated gene profiles

Table 2 Down-regulated genes in rats fed with WPH diet

Category and Gene Name Probe Set GB Accession No Fold Change P value

Cell adhesionEmbigin AJ009698 -657 0Cadherin 17 L46874 -48 0036Cadherin X78997 -336 0004Protein tyrosine phosphatase M60103 -264 0004Cytokeratin-8 S76054 -271 0Trans-Golgi network integral membrane protein TGN38 X53565 -492 0012Tumor-associated calcium signal transducer 1 AJ001044 -937 0001Claudin-3 AJ011656 -755 002Claudin-9 AJ011811 -512 0

Cell cyclegrowth controlMapk6 M64301 -261 0003Epithelial membrane protein 1 Z54212 -467 0015Glucagon K02813 -773 0005Peptide tyrosine-tyrosine (YY) M17523 -456 0001Src related tyrosine kinase U09583 -331 0033FGF receptor activating protein U57715 -425 0002Cyclin D1 D14014 -197 0001Neu oncogene X03362 -261 0017

Defenseimmunity proteinSeminal vesicle secretion protein iv J00791 -535 0001Putative cell surface antigen U89744 -522 0008Decay accelerating factor GPI AF039583 -612 0Beta defensin-1 AF093536 -2678 0001

DetoxificationantioxidationGlutathione S-transferase J02810 -517 0Glutathione S-transferase Yb X04229 -933 0

J03914 -243 0002Glutathione S-transferase alpha 1 K01932 -307 0002Glutathione transferase subunit 8 X62660 -642 0001Glutathione S-transferase Yc1 S72505 -369 0004Glutathione S-transferase Yc2 S72506 -2138 0008N-acetyltransferase 1 U01348 -464 0003Cytochrome P450CMF1b J02869 -823 0001Cytochrome P450 4F4 U39206 -643 0004Cytochrome P450 monooxygenase U39943 -282 0011Cytochrome P450 pseudogene U40004 -287 0Cytochrome P450 3A9 U46118 -691 0Cytochrome P450IVF M94548 -578 0002Cytochrome P450 subfamily 51 U17697 -207 0005Alcohol dehydrogenase M15327 -206 0025Aldehyde dehydrogenase M23995 -1056 0035

AF001898 -272 0004D-amino-acid oxidase AB003400 -1369 03-methylcholanthrene-inducible UDP-glucuronosyltransferase

S56937 -9 0

UDP-glucuronosyltransferase D38062 -317 0005D38065 -329 0002

UDP glycosyltransferase 1 D83796 -687 0J02612 -658 0J05132 -403 0

MetabolismMeprin 1 alpha S43408 -382 0014Brain serine protease bsp1 AJ005641 -442 0007Cystathionine gamma-lyase D17370 -305 0002Cathepsin S L03201 -262 0Meprin beta-subunit M88601 -5 0004Disintegrin and metalloprotease domain 7 X66140 -1191 0Fucosyltransferase 1 AB006137 -496 0001Fucosyltransferase 2 AB006138 -797 0017

UDP-glucoseceramide glycosyltransferase AF047707 -286 0007Type II Hexokinase D26393 -27 0001Hexokinase II S56464 -445 0007CDP-diacylglycerol synthase AB009999 -466 0Carboxylesterase precursor AB010635 -529 0002Fatty acid Coenzyme A ligase AB012933 -25 00413beta-HSD L17138 -327 011-beta-hydroxylsteroid dehydrogenase type 2 U22424 -3 0001Peroxiredoxin 6 AF014009 -355 001Platelet phospholipase A2 X51529 -325 0001

Ligand bindingcarrierCarnitine transporter AB017260 -395 0005Chloride channel (ClC-2) AF005720 -569 0002Putative potassium channel AF022819 -484 0Mitochondrial dicarboxylate carrier AJ223355 -355 0009Aquaporin 3 D17695 -783 0Na_H_Exchanger L11236 -981 0003Angiotensinvasopressin receptor (AIIAVP) M85183 -33 0002H+ K+-ATPase M90398 -1387 0Intestinal fatty acid binding protein K01180 -729 0001Apolipoprotein A-I precursor M00001 -345 0023Apolipoprotein A-I J02597 -247 0003Sodium-hydrogen exchange protein-isoform 3 M85300 -736 0004Liver fatty acid binding protein V01235 -262 0Sodium transporter X59677 -38 0Cation transporter X78855 -362 0003ATP-binding cassette AB010467 -389 0004Methionine adenosyltransferase II alpha J05571 -291 0007Phenylalanine hydroxylase M12337 -743 0Carbonic anhydrase IV S68245 -428 0011

Signal transductionB7 antigen X76697 -17095 0002CD24 antigen U49062 -308 0Chemokine CX3C AF030358 -504 0011Itmap1 AF022147 -75 0001HCNP E05646 -25 0001Brain glucose-transporter protein M13979 -297 0019Protein kinase C delta M18330 -248 0002Guanylate cyclase 2C M55636 -458 0003A2b-adenosine receptor M91466 -28 004Guanylate cyclase activator 2A M95493 -418 0005Phospholipase C beta-3 M99567 -257 0018Tm4sf3 Y13275 -333 0Phospholipase D AB000778 -271 0009BEM-2 D45413 -641 0015Sgk L01624 -393 0

Stress responseapoptosisProstaglandin D synthetase J04488 -4311 0009GTP cyclohydrolase I M58364 -326 0014

Structure proteinsChromogranin B (Chgb) AF019974 -256 0005Intestinal mucin M76740 -509 0002Muc3 U76551 -1107 0006Mucin-like protein M81920 -1197 0001Myosin 5B U60416 -394 0Keratin 18 X81448 -323 0004Keratin 19 X81449 -269 0001ZG-16p protein Z30584 -443 0002Plasmolipin Z49858 -72 0Cytokeratin 21 M63665 -496 0Syndecan S61865 -33 0006Claudin 3 M74067 -668 001

Table 2 Down-regulated genes in rats fed with WPH diet (Continued)

more down- than up-regulated genes were noted for bothSPI and WPH Additionally 37 genes were co-repressedwhereas only two were co-induced by SPI and WPH(Table 6) More than 90 of identified genes in WPH andSPI animals showed the same direction of change relativeto CAS This is visually apparent in the hierarchical clus-tering output (Fig 2)

Gene expression effects of WPHAs based on Gene Ontology (GO) annotations the 44 up-regulated and 119 down-regulated genes of the WPHgroup belong to multiple functional categories includingcell adhesion (n = 10) cell cycle and growth control (n =10) detoxification (n = 17) defense and immunity (n =7) signal transduction (n = 29) transcriptional regulation(n = 6) metabolism (n = 19) ligands and carriers (n =27) cell death (n = 3) structural proteins (n = 16) andothers (Tables 2 amp3) The fold change for up-regulatedgenes ranged between 21 [small nuclear ribonucleoparti-cle-associated protein (snRNP)] to 47 (argininosuccinatesynthetase) whereas down-regulated genes exhibited foldchanges between 20 (cyclin D1) and 171 (CD52antigen)

Lifetime ingestion of WPH affected the expression ofxenobiotic metabolism-related enzymes including severalof the cytochrome P450s and glutathione S-transferasesalcohol dehydrogenase (ADH) and UDP-glucuronosyl-transferase Cytochrome P450 enzymes and ADH are con-sidered to play key roles in activation of the proximatecarcinogen from AOM [29] Down-regulation of expres-sion of Phase I detoxification enzymes by WPH mighttherefore diminish AOM-induced DNA adducts andgenomic instability Consistent with results from a studyin which whey proteins inhibited cell proliferation in vitro[11] lifetime feeding of WPH was associated with changesin expression of genes involved in cell cycle control and

proliferation cyclin D1 neu oncogene mapk6 glucagonand peptide YY (PYY) genes were down-regulatedwhereas the expression of somatostatin a growth-inhibi-tory peptide was induced WPH altered expression ofgenes involved in cellular defense Induced genesincluded Ig gamma heavy chain adipsin and T-cell recep-tor beta chain whereas expression of the antibacterialpeptide beta defensin-1 and seminal vesicle secretion pro-tein IV (SVS IV) were down-regulated About 20 ofWPH-affected genes are involved in cell signaling theseinclude guanylate cyclase 2C protein kinase C delta andsynapsin Additionally genes encoding ligands or mem-brane channels [ie chloride channel intestinal fatty acidbinding protein (I-FABP) apoliprotein A-I (Apo-AI) Na+K+-ATPase and sodium transporter] were down-regulatedby WPH whereas calretinin and retinol binding protein(RBP) levels were increased

Gene expression effects of SPIColon genes whose mRNA expression was affected byingestion of SPI fell into multiple functional categoriesincluding cell adhesion (n = 4) cell cycle and growth con-trol (n = 6) detoxification (n = 18) defense and immu-nity (n = 6) signal transduction (n = 4) transcriptionalregulation (n = 1) metabolism (n = 8) ligands and carrierproteins (n = 17) cell death proteins (n = 5) and struc-tural proteins (n = 3) (Tables 4 amp5)

Relative abundance of numerous transcripts was changedin the same direction by WPH and SPI (Fig 2) Howeversome exceptions were noted For example mRNA encod-ing Apo-AI was down-regulated by WPH but elevated bySPI Apo-AI is the major determinant of the capacity ofHDL particles to promote cholesterol efflux and this pro-tein is associated with the inhibition of atherosclerosis[30] However the impact of differential response of Apo-AI to WPH and SPI on anti-tumorigenesis is unknown

Transcription factorregulatorHepatocyte nuclear factor 3 gamma AB017044 -696 0Apolipoprotein B mRNA editing protein L07114 -234 0DNA-binding inhibitor L23148 -41 001Kruppel-like factor 4 (gut) L26292 -308 0017

OthersProlactin receptor M74152 -326 0014LOC286964 U89280 -296 0003Ckmt1 X59737mRNA -265 0025Arginase II U90887 -2369 0Deleted in malignant brain tumors 1 U32681 -347 00023 end GAA-triplet repeat L13025 -273 0001Polymeric immunoglobulin receptor L13235 -293 0004

Changes in gene expression were determined by t-test (DMT) comparative analysis (MAS 50) and SAM (Stanford) Gene expression profiles from CAS animals were used as control P value and fold change are based on DMT analysis whereas final genes listed met all of the analytical criteria as described in Methods

Table 3 Up-regulated genes in rats fed with WPH diet

Category and Gene Name Probe Set GB Accession No

Fold Change P value

Cell adhesionFibronectin X05834 23 0EGF-containing fibulin-like extracellular matrix protein 1 D89730 217 0004

Cell cyclegrowth controlSomatostatin M25890 272 0001Somatostatin-14 K02248 387 0009APEG-1 U57097 324 0002

Defenseimmunity proteinIgG gamma heavy chain M28670 221 0009T-cell receptor beta chain X14319 214 0Adipsin M92059 321 0

Ligand bindingcarrierAngiotensin receptor M86912 275 0017Calretinin X66974 252 0005Purkinje cell protein 4 M24852 306 0001Secretogranin III U02983 277 0005Secretogranin II M93669 284 0001Aquaporin 1 X67948 34 0008Cacna2d1 M86621 284 0Retinol-binding protein M10934 217 0018

MetabolismLipoprotein lipase L03294 272 0Ubiquitin carboxyl-terminal hydrolase D10699 3 0003

Signal transductionThy-1 protein X02002 289 0002CD3 gamma-chain S79711 328 0002Synapsin M27925 394 0001Alpha-actinin-2 associated LIM protein AF002281 274 0009RESP18 L25633 274 0033T3 delta protein X53430 275 0003Protein phosphatase inhibitor-1 J05592t 26 0009CART protein U10071 216 0001Neuroendrocrine protein M63901 37 0006Protein kinase C-binding protein Zeta1 U63740 314 0003cannabinoid receptor 1 X55812 217 0002Guanylyl cyclase A J05677 318 0007Tachykinin 1 X56306 236 0036Protein tyrosine phosphatase L19180 247 0041Argininosuccinate synthetase X12459 469 0004

Stress responseapoptosisSmall inducible cytokine Y08358 335 0029

Structure proteinsFast myosin alkali light chain L00088 452 003Light molecular-weight neurofilament AF031880 241 0Neurofilament protein middle Z12152 297 0006Alpha-tubulin V01227 225 0Peripherin AF031878 282 0007

Transcription factorregulatorsnRNP M29293 211 0004snRNP-associated polypeptide X73411 333 0002

OthersC1-13 gene product X52817 317 0ND5 ND6 S46798 231 0015Sensory neuron synuclein X86789 284 0

Changes in gene expression were determined by t-test (DMT) comparative analysis (MAS 50) and SAM (Stanford) Gene expression profiles from CAS animals were used as control P value and fold change are based on the DMT analysis whereas listed genes met all of the analytical criteria as described in Methods

Table 4 Down-regulated genes in rats fed with SPI diet

Fold Change P value

Cell adhesionEmbigin AJ009698 -513 0001

Cell Cyclegrowth controlFGF receptor activating protein 1 U57715 -559 0002BEST5 protein Y07704 -237 0003Peptide tyrosine-tyrosine (YY) M17523 -391 0002Glucagon gene K02813 -658 0002Epithelial membrane protein-1 Z54212 -347 0017Neu oncogene X03362 -158 005

Defenseimmunity proteinBeta defensin-1 AF068860 -4216 0001

AF093536 -102 0Detoxificationantioxidation

Glutathione S-transferase J02810 -714 0Glutathione S-transferase Yb X04229 -1171 0001Glutathione S-transferase alpha 1 K01932 -418 0004Glutathione S-transferase Yc1 S72505 -523 0001Glutathione S-transferase Yc2 S72506 -527 0012

S82820 -345 0006Cytochrome P450 4F4 (CYP4F4) U39206 -652 0002Cytochrome P450CMF1b J02869 -412 0002Cytochrome P450 (CYP4F1) M94548 -288 00021-Cys peroxiredoxin Y17295 -255 0002Metallothionein M11794 -292 0006D-amino-acid oxidase AB003400 -542 0Peroxiredoxin 6 AF014009 -307 0008Phenylalanine hydroxylase M12337 -1099 0001

MetabolismDipeptidase L07315 -308 0001Meprin beta-subunit M88601 -327 0001Disintegrin and metalloprotease domain 7 X66140 -1403 0

Ligand bindingcarrierCarnitine transporter AB017260 -381 0003Chloride channel (ClC-2) AF005720 -326 0001Putative potassium channel AF022819 -269 0001Mitochondrial dicarboxylate carrier AJ223355 -254 001Aquaporin 3 D17695 -413 0Intestinal fatty acid binding protein K01180 -443 0005Na_H_Exchanger L11236 -447 0002H+ K+-ATPase M90398 -252 0001Carbonic anhydrase IV S68245 -428 0005Sodium transporter X59677 -34 0Phosphatidylethanolamine binding protein X75253 -269 0

Signal transductionB7 antigen X76697 -17095 0002HCNP E05646 -338 0Itmap1 AF022147 -797 0005Guanylate cyclase activator 2A M95493 -328 0006Sgk L01624 -276 0

Stress responseapoptosisProstaglandin D synthetase J04488 -458 001

Structure proteinsMuc3 U76551 -356 001Intestinal mucin M76740 -331 0006

Confirmation of differential gene expressionWe performed quantitative real-time RT-PCR on selectedgenes to confirm the microarray results Based uponknown associations with cell proliferation ordifferentiation 14 genes were chosen for further studyIncluded in this group was BTEB2 this gene was notpresent on the microarrays but was included in RT-PCRanalysis due to its significant expression in intestine andinvolvement in cell proliferation [see discussion] Asshown in Figure 3 eight genes were confirmed to be dif-ferentially expressed these included the gastrointestinalhormone genes PYY (129-fold down-regulated in WPHfed rats P = 0004) glucagon (178-fold down-regulatedin WPH fed rats P = 0005) and somatostatin (392-265-fold up-regulated in SPI and WPH fed rats P = 005P = 0025 respectively) cyclin D1 (16- 24-fold down-regulated in SPI and WPH fed rats P = 0033 P = 0001respectively) BTEB2 (19- 67-fold down-regulated in SPIand WPH fed rats P = 0024 P lt 0001 respectively) c-neu proto-oncogene (25- 41-fold down-regulated in SPIand WPH fed rats P lt 0001 P lt 0001 respectively) thecolonocyte differentiation marker I-FABP (29- 40-folddown-regulated in SPI and WPH fed rats P = 0023 P =001 respectively) and the mucin MUC3 (278- 405-fold down-regulated in SPI and WPH fed rats P lt 0001P lt 0001 respectively) Differential expression of fiveother genes was not confirmed statistically due to indi-vidual animal variation in the transcript levels howeverthe mean-fold changes for mRNA abundance were greaterthan two and in agreement with the corresponding micro-array results for these genes Only one of the selectedgenes ndash retinol binding protein (RBP) failed to exhibitgreater than a 2-fold change (in the predicted direction) atthe mRNA level by real-time RT-PCR

Serum somatostatin (Sst)As shown in Fig 4 circulating Sst concentration was sig-nificantly higher in rats fed WPH and SPI Colonic Sst pro-tein content in colon homogenates was below the limit ofdetection of the assay used (data not shown)

DiscussionThe type of dietary protein(s) can markedly affect theonset andor progression of CRC [31] Epidemiologicaland animal studies have found that dietary soy and wheyproteins decrease the incidence of certain tumors includ-ing those of the colon and rectum [6732-35] Using theAOM-treated male Sprague Dawley rat model we previ-ously found that lifetime feeding of SPI led to a ~ 76lower incidence of AOM-induced colon tumors comparedto rats lifetime-fed CAS [8] Additionally in the samestudies a ~ 46 lower incidence of colon tumors wasfound in WPH-fed compared to CAS-fed rats [9] Themolecular mechanism(s) by which these dietary proteinsreduce the incidence of chemically-induced colon tumorsis unclear although several mechanisms and putative bio-active factors have been proposed [11-24] The presentstudy has now identified genes that are differentiallyexpressed as a function of these diets and which serve tohighlight potential pathways for dietary protection fromcarcinogenesis

The ability to simultaneously analyze a large number ofdifferent mRNAs makes microarrays very appealing foridentifying genes and gene families whose expression isaltered by diet [3637] We focused on the normal colontissue since we are interested in genes that aredifferentially regulated by diet and which act in anti-onco-genic fashion in pre-cancerous tissues We limited ouranalysis to the proximal colon since several studies havesuggested that the molecular etiology of proximal and dis-tal colon tumors differs [2526] and proximal colontumors have become more prevalent with Westernizationof the diet and aging of the population [27] We chose toinclude colonic smooth muscle with the mucosa since a)the former tissue layer interacts with the latter to influenceits growth and function and b) we could monitor allcolonic genes affected by diet However one potentialcaveat to this strategy is the dilution effect that may havebeen imposed on the more rare mucosal transcriptsAnother caveat is that no information is obtained regard-ing where the differentially expressed transcripts occur In

Mucin-like protein M81920 -3 0Plasmolipin Z49858 -292 0003

Transcription factorregulatorTestis specific X-linked gene X99797 -691 0003

OthersArginase II U90887 -322 03-phosphoglycerate dehydrogenase X97772 -415 0017Aldehyde dehydrogenase family 1 AF001898 -393 0004

Changes in gene expression were determined by t-test (DMT) comparative analysis (MAS 50) and SAM (Stanford) Gene expression profiles from CAS animals were used as control P value and fold-change are based on the DMT analysis whereas genes listed above met all of the analytical criteria as described in Methods

Table 4 Down-regulated genes in rats fed with SPI diet (Continued)

this regard however we have confirmed by immuno-his-tochemistry that I-FABP is expressed predominantly in theinter-cryptal surface epithelium of colons from AOM-treated rats (Fig 5) Our study used a sample size of three(per diet group) which balanced the costs for the experi-mental reagents with the minimum number required forstatistical analysis The quantitative PCR analysesprovided confirmation that the filtering strategies usedyielded bona-fide differentially expressed transcripts

Only two transcripts were induced by both SPI and WPHwhereas 37 transcripts were repressed by both SPI andWPH This suggests that the cancer-protective actions ofthe two diets are generally associated with repression ofcolonic genes that facilitate tumorigenesis An alternativeexplanation is that CAS induces genes that facilitate coloncancer development when compared to SPI and WPH Itis also likely that SPI and WPH diets act in unique ways toinhibit tumorigenesis Irregardless our results indicatethat the nature of the dietary protein can profoundly affect

Table 5 Up-regulated genes in rats fed with SPI diet

Fold Change P value

Cell adhesionCollagen alpha1 type I Z78279 249 0Secreted phosphoprotein 1 M14656 11139 0006Matrix metalloproteinase 13 M60616 2434 0002Regenerating islet M62930 19308 0011

Defenseimmunity proteinIg gamma-2a chain L22654 11517 0001Ig gamma heavy chain M28670 322 0Ig germline kappa-chain C-region M18528 248 0038Ig light-chain U39609 263 0021Fc-gamma M32062 472 0017

DetoxificationGlutathione S-transferase 1 J03752 286 0Glutathione-S-transferasealpha type2 K00136 256 0009UDP glucuronosyltransferase D38066 283 0014

MetabolismMatrix metalloproteinase 7 L24374 363 002lysozyme rc_AA892775 277 0Matrix metalloproteinase 12 X98517 118 0013Mitochondrial carbamyl phosphate synthetase I M12335 5925 0001Aldolase B exon 9 X02291 87 001Aldolase B exon 2 X02284 271 0001

Signal transductionMHC class II antigen RT1B-1 beta-chain X56596 255 0001CD3 gamma-chain S79711 451 0001

Ligand bindingcarrierIntracellular calcium-binding protein L18948 2829 0014Retinol binding protein II M13949 511 0001Apolipoprotein B M27440 647 0024Apolipoprotein A-I J02597 249 0004Iron ion transporter AF008439 1878 0008

Stress responseapoptosisHeme oxygenase J02722 966 0002JE product X17053 352 0001Pancreatitis-associated protein M98049 6839 0004Pancreatitis associated protein III L20869 1535 0Reg protein E01983 3025 0001

OthersHistamine N-tele-methyltransferase S82579 617 004

Changes in gene expression were determined by t-test (DMT) comparative analysis (MAS 50) and SAM (Stanford) Gene expression profiles from CAS animals were used as control P value and fold-change are based on the DMT analysis whereas final listed genes met all of the analytical criteria as described in the Methods

colon gene expression profiles Thus gene expression pro-filing studies of colons should account for potential con-founding effects of diet

Dietary factors in SPI or WPH inhibit cell proliferationand induce apoptosis among other biological actions[1113] In the present study we identified cyclin D1 geneand the neu proto-oncogene as being repressed in proxi-mal colon by SPI and WPH Cyclin D1 is a key regulatorof cell cycle progression [38] and a target of β-catenin a

protein whose abnormal accumulation in the nucleus isstrongly linked to the development of multiple tumortypes including those of the colon [39] Aberrantlyincreased expression of cyclin D1 in colon epithelial cellscontributes to their abnormal proliferation and tumori-genicity [4041] Similarly the oncogenic and cellulargrowth-promoting activities of the HER-2neu proto-oncogene are well known [42] HER-2neu a tyrosinekinase receptor for neu-differentiation factor is expressedin normal colonic epithelium and is up-regulated in ade-

Table 6 Genes co-regulated with WPH and SPI diet

Category and Gene Name Probe Set GB Accession No Fold Change in WPH P value Fold Change in SPI P value

Down-regulated genesEmbigin AJ009698 -657 0 -513 0001Epithelial membrane protein 1 Z54212 -467 0015 -347 0017Glucagon K02813 -773 0005 -658 0002Peptide tyrosine-tyrosine (YY) M17523 -456 0001 -391 0002FGF receptor activating protein U57715 -425 0002 -559 0002Neu oncogene X03362 -261 0017 -158 005CD52 antigen X76697 -17095 0002 -17095 0002Beta defensin-1 AF068860 -5448 0001 -4216 0001Glutathione S-transferase J02810 -517 0 -714 0Glutathione S-transferase Yb X04229 -933 0 -1171 0001Glutathione S-transferase alpha 1 K01932 -307 0002 -418 0004Glutathione S-transferase Yc1 S72505 -369 0004 -523 0001Glutathione S-transferase Yc2 S72506 -2138 0008 -527 0012Cytochrome P450CMF1b J02869 -823 0001 -412 0002Cytochrome P450 4F4 U39206 -643 0004 -652 0002Cytochrome P450IVF M94548 -578 0002 -288 0002D-amino-acid oxidase AB003400 -1369 0 -542 0Meprin beta-subunit M88601 -5 0004 -327 0001Disintegrin and metalloprotease domain 7 X66140 -1191 0 -1403 0Carnitine transporter AB017260 -395 0005 -381 0003Chloride channel (ClC-2) AF005720 -569 0002 -326 0001Putative potassium channel AF022819 -484 0 -269 0001Mitochondrial dicarboxylate carrier AJ223355 -355 0009 -254 001Aquaporin 3 D17695 -783 0 -413 0Na_H_Exchanger L11236 -981 0003 -447 0002H+ K+-ATPase M90398 -1387 0 -252 0001Fatty acid binding protein 1 K01180 -729 0001 -443 0005Sodium transporter X59677 -38 0 -34 0Carbonic anhydrase IV S68245 -428 0011 -428 0005Itmap1 AF022147 -75 0001 -797 0005HCNP E05646 -25 0001 -338 0Guanylate cyclase activator 2A M95493 -418 0005 -328 0006Sgk L01624 -393 0 -276 0Prostaglandin D synthetase J04488 -4311 0009 -458 001Mucin 3 M76740 -509 0002 -331 0006Mucin-like protein M81920 -1197 0001 -3 0Plasmolipin Z49858 -72 0 -292 0003

Up-regulated genesIg gamma heavy chain M28670 221 0009 322 0CD3 gamma-chain S79711 328 0002 451 0001

Changes in gene expression were determined by t-test (DMT) comparative analysis (MAS 50) and SAM (Stanford) Gene expression profiles from CAS animals were used as control P value and fold change are based on the DMT analysis whereas final listed genes met all of the analytical criteria described in Methods

nomatous polyps of the colon [43] The down regulationof cyclin D1 and c-neu mRNA abundance by SPI andWPH may at least partly explain their anti-tumorigenicproperties Similarly Kruumlppel-like transcription factors

have been linked to cell growth and tumorigenesis BTEB2(also known as Kruumlppel-like factor 5 KLF5 or intestinalKLF) was reported to enhance intestinal epithelia cell col-ony formation cyclin D1 transcription and cell prolifera-tion [44] Consistent with our results for cyclin D1colonic BTEB2 mRNA expression was down-regulated bySPI and WPH Aquaporin 3 a water channel highlyexpressed in colonic epithelium was down-regulated bySPI and WPH Aquaporins are thought to be induced earlyin colon cancer and to facilitate oncogenesis [45]therefore dietary repression of such genes may addition-ally contribute to anti-tumorigenesis The results for I-FABP and MUC3 indicated 3ndash4 fold decreases in tran-

Quantitative real-time RT-PCR verification of microarray resultsFigure 3Quantitative real-time RT-PCR verification of microarray results RNA used for real-time RT-PCRs was from the same animals (n = 7 per diet group) whose RNAs comprised the pools for microarray analysis Values are mean plusmn SEM and were analyzed by one-way ANOVA P lt 005 SPI or WPH vs CAS

14 CASWPHSPI

Neu Oncogene Cyclin D1 BTEB2

16 CASWPHSPI

18 CASWPHSPI

Somatostatin Peptide YY Glucagon

BTEB1 Muc3 I-FABP

Immuno-histochemistry for I-FABP in colons from AOM-treated ratsFigure 5Immuno-histochemistry for I-FABP in colons from AOM-treated rats Panels A and B are sections from CAS and WPH-fed animals respectively Arrows point to the strong areas of staining for I-FABP in the inter-cryptal surface epi-thelium (overall intensity of staining is greater for CAS than for WPH)

script abundance in proximal colons of rats on SPI orWPH diets These particular results are not easilyreconciled with decreased tumorigenesis in SPI and WPHgroups since both genes are highly expressed in the nor-mal differentiated colonic epithelium and are likely to beunder-expressed in adenomas and adenocarcinomas [46]Perhaps these represent diet-modulated genes that arenot direct participants in anti-tumorigenesis

Gastrointestinal hormones regulate a myriad of intestinalfunctions including motility absorption digestion cellproliferation and death and immune response [47] Themicroarray and real-time RT-PCR assays identified induc-tive effects of SPI and WPH on somatostatin mRNA andprotein abundance These results implicate this geneproduct in autocrine and paracrine mechanisms underly-ing colon cancer protection by SPI and WPH sincesomatostatin is a well-known anti-proliferative agent forcolon tumor cells [4849] This hormone is also a negativeregulator of angiogenesis [50] this is predicted to countertumorigenesis It is possible that the small decrements inrat growth rates observed with lifetime SPI or WPH diets[89] are a consequence of this increased circulating soma-tostatin level We also found decreased abundance ofmRNAs encoding peptide YY (PYY) and glucagon incolons of WPH-fed rats PYY gene expression in humancolon tumors is much reduced relative to the adjacentnormal tissue [51] however chemically-induced colontumors in rats generally exhibit higher overall expressionof PYY due to increased prevalence of PYY-positive cellscompared to normal mucosa [5253] PYY stimulates pro-liferation of intestinal epithelium [54] therefore an inhi-bition of PYY expression by dietary WPH may contribute

to colon cancer-protective actions A similar scenariomight apply to colon glucagon gene expression as thisgrowth stimulatory peptide for colon cancer cells [55] wasinhibited by WPH at the level of colon mRNA abundance

Our data highlighted other aspects of diet and colon geneexpression that warrant further study For example the B7antigen (also known as CD52) mRNA was strongly down-regulated by SPI or WPH The corresponding protein isnormally expressed at high levels on cell membranes of Tand B lymphocytes and monocytes infusion with anti-CD52 antibody leads to systemic depletion of T cells [56]The lower abundance of this transcript in non-tumorcolon tissue of rats on SPI or WPH diets may reflect fewernumbers of immune cells in this tissue as compared toCAS-fed animals One possible interpretation of this datais that the normal tissue of the CAS group has manifestedan immune response perhaps in response to increasedtumorigenicity relative to SPI or WPH groups Such aninterpretation raises the prospect of a functional immuno-editing mechanism [57] occurring in this model of coloncancer and an indirect effect of diet on lymphocyte popu-lations (via presence of tumors or tumor precursors) inthe colon An alternative mechanism is that dietaryprotein can directly affect the populations of lymphocytesresident in the colon which in turn may affect tumori-genesis A related observation was the enhanced abun-dance of CD3 gamma chain transcripts in colons of SPIand WPH animals The protein encoded by this transcripthelps mediate T cell antigen receptor engagement and sig-naling [58] its decreased abundance in colonic T cells ofCAS-fed animals may indicate a specific immune defect[59] occurring in the CAS-fed animals after exposure tocarcinogen and thereby contributing to enhanced tumor-igenesis in this group

Several microarray studies of human paired normal colonvs colon tumors have been published [60-64] Compari-son of the present results for normal colon tissue of AOM-treated rats on different diets to the published studies forhuman CRC identified only a small number of commondifferentially expressed genes andor gene families incommon (data not shown) This small number is proba-bly due to the fact that our study did not examine colontumors rather we focused on normal colon tissue In thisregard it will be interesting to examine the expressionprofiles of colons from animals not treated with AOM soas to more specifically correlate transcripts with diet andcancer phenotype This study has illuminated a number ofgenes and gene families that may act as dietary protein-dependent modulators of oncogenesis in the rat colonAdditional studies that specifically address the functionalinvolvement of these genes in cancer-prevention via die-tary means are required to confirm the postulated roles

Diet effects on serum Sst concentrationFigure 4Diet effects on serum Sst concentration Values are mean plusmn SEM One-way ANOVA P lt 005 SPI or WPH vs CAS

P=0005

P=0011

CAS WPH SPI

ConclusionsWe have identified genes in rat colon that are differen-tially expressed as a consequence of altered dietary pro-tein during AOM-induced oncogenesis These arecandidates for genes that sub-serve the anti-cancer effectsof dietary SPI and WPH in this tissue

MethodsRats diets and carcinogen treatmentThe animals whose colons were used in the present studyhave been previously described [89] Time-mated [gesta-tion day (GD) 4] Sprague-Dawley rats were purchasedfrom Harlan Industries (Indianapolis IN) housedindividually and allowed ad libitum access to water andpelleted food Rats were randomly assigned to one ofthree semi-purified isocaloric diets made according to theAIN-93G diet formula [65] and which differed only byprotein source a) CAS (New Zealand Milk Products SantaRosa CA) b) WPH (New Zealand Milk Products SantaRosa CA) or c) SPI (Dupont Protein Technologies StLouis MO) Offspring were weaned to the same diet astheir mothers and were fed the same diets throughout thestudy At 90 days of age male offspring received sc injec-tions of 15 mgkg AOM (Ash Stevens Detroit MI) insaline once a week for 2 weeks Forty weeks later rats wereeuthanized and the colon (cecum to anus) was dividedinto two equal segments (proximal and distal) openedlongitudinally and examined for tumors We found thatboth WPH and SPI significantly decreased the colontumor incidence [data published in [89]] Arepresentative non-tumor segment of each proximalcolon (PC) was frozen in liquid nitrogen and stored at -80degC for later use Animal care and handling were inaccordance with the Institutional Animal Care amp UseCommittee guidelines of the University of Arkansas forMedical Sciences

RNA processingTotal RNA was isolated from rat proximal colons (n = 7for each of CAS SPI and WPH diets) using TRIzol reagent(Invitrogen Carlsbad CA) and further purified with theRNeasy Mini Kit (QIAGEN Valencia CA) To remove con-taminating DNA on-column DNA digestion with RNase-Free DNase (QIAGEN) was performed Integrity of iso-lated RNAs was confirmed using the RNA 6000 NanoLabChip kit with the Agilent 2100 Bioanalyzer System(Agilent Biotechnologies Palo Alto CA) To reduce errorsdue to biological variability RNA samples were pooled asproposed by Bakay et al [66] Pooled RNA (equal amountsof RNA from each of 7 animals 8 ug total) was used forcDNA synthesis using a T7-(deoxythymidine)24 primerand Superscript II (Life Technologies Inc GaithersburgMD) The resulting cDNA was used with the ENZO BioAr-ray High Yield RNA Transcript labeling kit (ENZOFarmingdale NY) to synthesize biotin-labeled cRNA The

cRNA was purified on RNeasy spin columns (QIAGEN)and subjected to chemical fragmentation (size range of 35to 200 bp) Three replicate cRNA targets were made in par-allel starting from each RNA pool

Microarray proceduresTen ug of cRNA was hybridized for 16 hours to an Affyme-trix (Santa Clara CA) rat U34A GeneChip (3 chips usedper diet group) followed by incubations with streptavi-din-conjugated phycoerythrin and then with polyclonalanti-streptavidin antibody coupled to phycoerythrin Fol-lowing washing GeneChips were scanned using an Agi-lent GeneArray laser scanner Images were analyzed usingAffymetrix MAS 50 software Bacterial sequence-derivedprobes on the arrays served as external controls for hybrid-ization whereas the housekeeping genes β-actin andGAPDH served as endogenous controls and for monitor-ing the quality of the RNA target To compare array databetween GeneChips we scaled the average of thefluorescent intensities of all probes on each array to a con-stant target intensity of 500

BioinformaticsTo validate the microarray procedure for our samplesunsupervised nearest-neighbor hierarchical clustering(Spotfire Somerville MA) was performed on gene expres-sion data The inter-chip variability test also was per-formed as specified in the Affymetrix data analysis manual[28] To identify colon genes differentially expressed withSPI or WPH (control CAS diet) multiple criteria wereapplied final results are reported only for transcripts thatpassed all three analytical steps described below Firstlythe t-test feature of DMT (Affymetrix) was used to identifygenes whose expression was regulated (inducedrepressedwith P lt 005) by SPI or WPH and signal fold changes(FC) for these genes were calculated Secondly microarraydata were analyzed using Significance of Analysis ofMicroarrays (SAM Stanford) to identify significantchanges in gene expression among diet groups [67] usinga false discovery rate (FDR) cutoff of 05 Lastly a pair-wise comparison survival (3 times 3) method was used toidentify differentially expressed transcripts [68] In briefthe three replicate expression profiles obtained for SPIcolons were iteratively compared with the three CAS pro-files (latter as baseline) in MAS 50 (Affymetrix) generat-ing nine comparisons in total Transcripts with a log ratiogreater than or equal to 1 (ge2 fold change) whichincreased (I) in nine of nine comparisons and which wereexpressed above background (ie called as Present) in allthree SPI GeneChips were considered to be up-regulatedby SPI Transcripts with a log ratio less than or equal to -1 were decreased (D) in nine of nine comparisons andexpressed above background (Present) in all three CASchips were considered to be down-regulated by SPI WPH-regulated genes were similarly identified Genes that were

independently identified by all three approaches com-prised the final reported lists of differentially expressedgenes (Tables 2 3 4 5 6)

Validation of gene expression by quantitative real-time RT-PCROne microg of total RNA from each of the 21 individual prox-imal colons (which comprised the original pools for themicroarray experiment) was reverse-transcribed using ran-dom hexamers and MultiScribe Reverse Transcriptase in atwo-step RT-PCR reaction (Applied Biosystems FosterCity CA) Primers (Table 7) were designed using PrimerExpress (Applied Biosystems) and were selected to yield asingle amplicon this was verified by dissociation curvesandor analysis in agarose gels SYBR Green real-time PCRwas performed with an ABI Prism 7000 Sequence Detec-tor Thermal cycling conditions included pre-incubationat 50degC for 2 min 95degC for 10 min followed by 40 PCRcycles at 95degC for 15 sec and 60degC for 1 min The relativetranscript levels for each gene were calculated using therelative standard curve method (User Bulletin 2 AppliedBiosystems) and normalized to the house-keeping gene β-actin Data are reported as mean plusmn SEM of n = 7 animalsper dietary group Significant differences between dietgroups were determined by one-way ANOVA (P lt 005)

Serum SstRat serum Sst content (15 animals from each diet) wasdetermined using the somatostatin-28 EIA kit purchasedfrom Phoenix Pharmaceuticals Corporation (BelmontCalifornia)

Authors contributionsRX performed the microarray and real-time PCR experi-ments conducted the data analysis and participated indrafting the manuscript TMB designed and oversaw theanimal component of the study FAS designed the analyt-ical and overall approaches to the study supervised theproject and drafted the manuscript All authors read andapproved the final manuscript

AcknowledgementsWe thank Dr Rosalia CM Simmen and Dr Rick Helm for insightful com-ments on the manuscript and Amanda L Linz for performing the I-FABP immuno-histochemistry Supported by USDA CRIS 6251-5100-002-06S

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Table 7 Primer sequences for real-time RT-PCR

Gene Forward primer Reverse primer Accession no

Beta-actin 5-GACGGTCAGGTCATCACTATCG-3 5-ACGGATGTCAACGTCACACTTC-3 NM_031144I-FABP 5-AGGAAGCTTGGAGCTCATGACA-3 5-TCCTTCCTGTGTGATCGTCAGTT-3 K01180Neu Oncogene 5-GTGGTCGTTGGAATCCTAATCAA-3 5-CCTTCCTTAGCTCCGTCTCTTTTA-3 X03362PYY 5-AGGAGCTGAGCCGCTACTATGC-3 5-TTCTCGCTGTCGTCTGTGAAGA-3 M17523Glucagon 5-TGGTGAAAGGCCGAGGAAG-3 5-TGGTGGCAAGGTTATCGAGAA-3 K02813Somatostatin 5-GGAAACAGGAACTGGCCAAGT-3 5-TGCAGCTCCAGCCTCATCTC-3 K02248PAP III 5-AAGAGGCCATCAGGACACCTT-3 5-CACTCCCATCCACCTCTGTTG-3 L20869CYP4F1 5-CCAAGTGGAAACGGTTGATTTC-3 5-TCCTGGCAGTTGCTGTCAAAG-3 M94548GST 5-ACTTCCCCAATCTGCCCTACTTA-3 5-CGAATCCGCTCCTCCTCTGT-3 X04229Cyclin D1 5-TCAAGTGTGACCCGGACTGC-3 5-ACTTCCCCTTCCTCCTCGGT-3 D14014Beta defensin-1 5-TCTTGGACGCAGAACAGATCAATA-3 5-TCCTGCAACAGTTGGGCTATC-3 AF093536H+ K+-ATPase 5-ATTCCGCATCCCTAGACAACG-3 5-TCTTACTAAAGCTGGCCATGATGTT-3 M90398Prostaglandin D synthetase 5-CAAGCTGGTTCCGGGAGAAG-3 5-TTGGTCTCACACTGGTTTTTCCTTA-3 J04488RBP 5-TCGTTTCTCTGGGCTCTGGTAT- 3 5-TTCCCAGTTGCTCAGAAGACG-3 M10934Muc3 5-AAGGTGTGAGGAAGTGATGGAGA-3 5-GCAGAGACCGTCGGCTTTATC-3 U76551BTEB1 5-ACACTGGTCACCATCGCCAA-3 5-GGACTCGACCCAGATTCGGT-3 NM_057211BTEB2 5-CTACTTTCCCCCATCACCACC-3 5-GAATCGCCAGTTTCGAAGCA-3 AB096709

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Abstract

Background

Results

Conclusions

Background

Results

Validation of the microarray approach

Table 2

Differentially expressed genes

Gene expression effects of WPH

Gene expression effects of SPI

Table 3

Table 4

Confirmation of differential gene expression

Serum somatostatin (Sst)

Discussion

Table 6

Conclusions

Methods

Rats diets and carcinogen treatment

RNA processing

Microarray procedures

Bioinformatics

Validation of gene expression by quantitative real-time RT-PCR

Serum Sst

Authors contributions

Acknowledgements

References

BackgroundColorectal cancer (CRC) is the third most common cancerand the third leading cause of cancer-related mortality inthe US [12] Estimated new cases of colon cancer were79650 for men and 73530 for women in 2004 [1]approximately $63 billion is spent in the United Stateseach year on treatment of CRC [2] Accumulating evi-dence suggests that diet is an important environmentalfactor in the etiology of CRC High consumption of redmeats animal fats chocolate alcohol and refined cerealsare linked to higher incidence of these cancers in Westernsocieties [3-5] whereas protective effects of fruits vegeta-bles and whole grains have been suggested [5]

Soy foods and soybean constituents have received consid-erable attention for their potential role in reducing cancerrisk [67] Our laboratories reported the protective effectsof lifetime ingestion of soy protein isolate (SPI) onazoxymethane (AOM)-induced colon cancer in rats [8]Similarly the effect of whey protein hydrolysate (WPH) inthe diet to reduce colon tumor incidence has beenreported by us and others [9-11] Several hypotheses havebeen proposed to account for soy and whey protein-induced anti-tumorigenesis For example soy isoflavoneshave been proposed to play a key role in soys anti-cancerfunctions [12] Yanagihara et al among others reportedthat genistein inhibits colon cancer cell proliferation andstimulates apoptosis in vitro [13-15] However subcuta-neous administration of genistein to mice did not confirmthese in vitro effects [16] Holly et al reported that soysphingolipids inhibit colonic cell proliferation and sug-gested that this may partially account for its anticancerbenefits [17] Other reports indicate that soy diets inhibittumorigenesis by regulating the synthesis or activities ofspecific proteins For example Rowlands et al reportedthat dietary soy and whey proteins down-regulate expres-sion of liver and mammary gland phase I enzymesinvolved in carcinogen activation [18] Elevated activitiesof phase II detoxification enzymes were reported in soy-fed rats [1920] Such dietary effects may result in lowertissue concentrations of activated carcinogen The antican-cer properties of whey proteins have been ascribed to theirability to elevate cellular levels of the antioxidant glutath-ione [2122] Moreover the whey protein α-lactalbumininhibits proliferation of mammary epithelial cells in vitro[11] The anticancer properties of whey may also relate toits immune system-enhancing actions [23]

Despite extensive research there is no consensus for anti-cancer mechanism(s) of soy and whey which willundoubtedly involve multiple interrelated processespathways and many components Many of the samemolecular and biochemical changes underlying humancolon cancer are observed in the azoxymethane (AOM)-induced rat colon cancer model [24] Moreover previous

studies suggest a different molecular etiology for tumorsof the proximal and distal colon in this model and inhuman colon [2425] Differential dietary effects on prox-imal vs distal colon DNA damage were noted [26] andWesternization of the human diet is thought to havefavored a shift of tumors from distal to more proximallocations [27] Thus region-specific localization of dietaryeffects on colon tumorigenesis is an important factor toconsider in any molecular analysis of CRC Here we useAffymetrix high-density oligonucleotide microarrays todetermine the expression profiles of non-tumor (ie nor-mal) tissue in proximal colons (PC) of rats subjected tolifetime diets containing casein (CAS control diet) soyprotein isolate (SPI) or whey protein hydrolysate (WPH)and which were administered AOM to induce tumors Wehypothesized that genes whose expression contributes toanti-tumorigenesis would be regulated in parallel by SPIand WPH in addition changes unique to each diet mightalso be apparent

ResultsValidation of the microarray approachQuality control steps ensured that the RNA used formicroarray and real-time RT-PCR analysis was of highquality These steps included evaluation of the RNA withthe RNA 6000 Nano Assay and assessment of the cRNAhybridization to GeneChips by comparison of dataobtained for probe sets representative of 5 and 3 ends ofcontrol genes All RNA samples had an A260280 absorb-ance ratio between 19 and 21 The ratio of 28S to 18SrRNA was very close to 2 on RNA electropherograms andsignal ratios below 3 were noted for 3 vs 5 probe sets forβ-actin and glyceraldehyde-3-phosphate dehydrogenase(per Affymetrix user guidelines) after hybridization

Total false change rates (TFC) were determined followingAffymetrix-recommended guidelines [28] except that theinter-chip comparisons used cRNA targets made in paral-lel starting from the same RNA pool Inter-chip variabilitymeasured as TFC was 025 ndash 06 and well below thesuggested 2 cutoff (Table 1) These values confirmed thefidelity and reproducibility of the microarray procedures

Table 1 Inter-chip variability

Diet group Number of arrays TFC ()

CAS 3 0252 plusmn 0138WPH 3 0369 plusmn 0025SPI 3 0570 plusmn 0165

TFC (Total false change) = false change rate (decreased category) + false change rate (increased category) as described in ref 28 TFC reported as mean plusmn SEM TFC should be no more than 2 (Affymetrix)

S56937 -9 0

Fold Change P value

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

Results

Conclusions

Background

Results


Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

S56937 -9 0

Fold Change P value

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

Results

Conclusions

Background

Results


Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

S56937 -9 0

Fold Change P value

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

Results

Conclusions

Background

Results


Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

Fold Change P value

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

Results

Conclusions

Background

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Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

Fold Change P value

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

Results

Conclusions

Background

Results


Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

Fold Change P value

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

Results

Conclusions

Background

Results


Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

Fold Change P value

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

Results

Conclusions

Background

Results


Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

Fold Change P value

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

Results

Conclusions

Background

Results


Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

Fold Change P value

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

Results

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Background

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Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

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Background

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Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

14 CASWPHSPI

16 CASWPHSPI

18 CASWPHSPI

BTEB1 Muc3 I-FABP

P=0005

P=0011

CAS WPH SPI

BioMedcentral

Abstract

Background

Results

Conclusions

Background

Results


Table 2




Table 3

Table 4



Discussion

Table 6

Conclusions

Methods


RNA processing


Bioinformatics


Serum Sst


Acknowledgements

References

P=0005

P=0011

CAS WPH SPI

BioMedcentral

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Table 2




Table 3

Table 4



Discussion

Table 6

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RNA processing


Bioinformatics


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BioMedcentral

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BioMedcentral

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BioMedcentral

Abstract

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BioMedcentral

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Documents

Molecular Cancer BioMed Central - COnnecting REpositories · data set so as to identify differentially expressed colon transcripts in rats fed SPI, WPH or CAS; results are reported