Dietary exposure to soy or whey proteins alters colonic global gene expression profiles during rat colon tumorigenesis

1476-4598-4-1 1476-4598 Research Dietary exposure to soy or whey proteins alters colonic global gene expression profiles during rat colon tumorigenesis Xiao Rijin XiaoRijin@uams.edu Badger M Thomas BadgerThomasM@uams.edu Simmen A Frank SimmenFrankA@uams.edu

Arkansas Children's Nutrition Center, 1120 Marshall Street, Little Rock, AR, 72202, USA

Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR, 72202, USA

Molecular Cancer 1476-4598 2005 4 1 1 http://www.molecular-cancer.com/content/4/1/1 15644144 10.1186/1476-4598-4-1 08 9 2004 11 1 2005 11 1 2005 2005 Xiao et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. colon cancer soy whey gene expression profiling neuro-endocrine microarray rat

Abstract

Background

We previously reported that lifetime consumption of soy proteins or whey proteins reduced the incidence of azoxymethane (AOM)-induced colon tumors in rats. To obtain insights into these effects, global gene expression profiles of colons from rats with lifetime ingestion of casein (CAS, control diet), soy protein isolate (SPI), and whey protein hydrolysate (WPH) diets were determined.

Results

Male Sprague Dawley rats, fed one of the three purified diets, were studied at 40 weeks after AOM injection and when tumors had developed in some animals of each group. Total RNA, purified from non-tumor tissue within the proximal half of each colon, was used to prepare biotinylated probes, which were hybridized to Affymetrix RG_U34A rat microarrays containing probes sets for 8799 rat genes. Microarray data were analyzed using DMT (Affymetrix), SAM (Stanford) and pair-wise comparisons. Differentially expressed genes (SPI and/or 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 group, relative to CAS. Hierarchical clustering identified the co-induction or co-repression of multiple genes by SPI and WPH. The differential expression of I-FABP (2.92-, 3.97-fold down-regulated in SPI and WPH fed rats; P = 0.023, P = 0.01, respectively), cyclin D1 (1.61-, 2.42-fold down-regulated in SPI and WPH fed rats; P = 0.033, P = 0.001, respectively), and the c-neu proto-oncogene (2.46-, 4.10-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively) mRNAs were confirmed by real-time quantitative RT-PCR. SPI and WPH affected colonic neuro-endocrine gene expression: peptide YY (PYY) and glucagon mRNAs were down-regulated in WPH fed rats, whereas somatostatin mRNA and corresponding circulating protein levels, were enhanced by SPI and WPH.

Conclusions

The identification of transcripts co- or differentially-regulated by SPI and WPH diets suggests common as well as unique anti-tumorigenesis mechanisms of action which may involve growth factor, neuroendocrine and immune system genes. SPI and WPH induction of somatostatin, a known anti-proliferative agent for colon cancer cells, would inhibit tumorigenesis.

Background

Colorectal cancer (CRC) is the third most common cancer and the third leading cause of cancer-related mortality in the U.S. 12. Estimated new cases of colon cancer were 79,650 for men and 73,530 for women in 2004 1; approximately $6.3 billion is spent in the United States each year on treatment of CRC 2. Accumulating evidence suggests that diet is an important environmental factor in the etiology of CRC. High consumption of red meats, animal fats, chocolate, alcohol and refined cereals are linked to higher incidence of these cancers in Western societies 345, whereas protective effects of fruits, vegetables and whole grains have been suggested 5.

Soy foods and soybean constituents have received considerable attention for their potential role in reducing cancer risk 67. Our laboratories reported the protective effects of lifetime ingestion of soy protein isolate (SPI) on azoxymethane (AOM)-induced colon cancer in rats 8. Similarly, the effect of whey protein hydrolysate (WPH) in the diet to reduce colon tumor incidence has been reported by us and others 91011. Several hypotheses have been proposed to account for soy and whey protein-induced anti-tumorigenesis. For example, soy isoflavones have been proposed to play a key role in soy's anti-cancer functions 12. Yanagihara et al., among others, reported that genistein inhibits colon cancer cell proliferation and stimulates apoptosis in vitro 131415. However, subcutaneous administration of genistein to mice did not confirm these in vitro effects 16. Holly et al. reported that soy sphingolipids inhibit colonic cell proliferation, and suggested that this may partially account for its anticancer benefits 17. Other reports indicate that soy diets inhibit tumorigenesis by regulating the synthesis or activities of specific proteins. For example, Rowlands et al. reported that dietary soy and whey proteins down-regulate expression of liver and mammary gland phase I enzymes involved in carcinogen activation 18. Elevated activities of phase II detoxification enzymes were reported in soy-fed rats 1920. Such dietary effects may result in lower tissue concentrations of activated carcinogen. The anticancer properties of whey proteins have been ascribed to their ability to elevate cellular levels of the antioxidant glutathione 2122. Moreover, the whey protein, α-lactalbumin, inhibits proliferation of mammary epithelial cells in vitro 11. The anticancer properties of whey may also relate to its immune system-enhancing actions 23.

Despite extensive research, there is no consensus for anti-cancer mechanism(s) of soy and whey, which will undoubtedly involve multiple interrelated processes, pathways and many components. Many of the same molecular and biochemical changes underlying human colon cancer are observed in the azoxymethane (AOM)-induced rat colon cancer model 24. Moreover, previous studies suggest a different molecular etiology for tumors of the proximal and distal colon in this model and in human colon 2425. Differential dietary effects on proximal vs. distal colon DNA damage were noted 26 and Westernization of the human diet is thought to have favored a shift of tumors from distal to more proximal locations 27. Thus, region-specific localization of dietary effects on colon tumorigenesis is an important factor to consider in any molecular analysis of CRC. Here, we use Affymetrix high-density oligonucleotide microarrays to determine the expression profiles of non-tumor (i.e., normal) tissue in proximal colons (PC) of rats, subjected to lifetime diets containing casein (CAS, control diet), soy protein isolate (SPI), or whey protein hydrolysate (WPH) and which were administered AOM to induce tumors. We hypothesized that genes whose expression contributes to anti-tumorigenesis would be regulated in parallel by SPI and WPH; in addition, changes unique to each diet might also be apparent.

Results

Validation of the microarray approach

Quality control steps ensured that the RNA used for microarray and real-time RT-PCR analysis was of high quality. These steps included evaluation of the RNA with the RNA 6000 Nano Assay and assessment of the cRNA hybridization to GeneChips by comparison of data obtained for probe sets representative of 5' and 3' ends of control genes. All RNA samples had an A260/280 absorbance ratio between 1.9 and 2.1. The ratio of 28S to 18S rRNA was very close to 2 on RNA electropherograms, and signal ratios below 3 were noted for 3' vs. 5' probe sets for β-actin and glyceraldehyde-3-phosphate dehydrogenase (per Affymetrix user guidelines) after hybridization.

Total false change rates (TFC) were determined following Affymetrix-recommended guidelines 28, except that the inter-chip comparisons used cRNA targets made in parallel starting from the same RNA pool. Inter-chip variability, measured as TFC%, was 0.25% – 0.6% and well below the suggested 2% cutoff (Table 1). These values confirmed the fidelity and reproducibility of the microarray procedures used. Unsupervised nearest-neighbor hierarchical clustering identified differences in proximal colon gene expression 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 tissue of AOM-treated rats. Interestingly, the overall gene expression profiles for SPI and WPH groups were more similar to each other than each was to the CAS group (Fig. 1A).

Table 1

Inter-chip variability

Diet group

Number of arrays

TFC (%)*

CAS

0.252 ± 0.138

WPH

0.369 ± 0.025

SPI

0.570 ± 0.165

*TFC (Total false change) = false change rate (decreased category) + false change rate (increased category), as described in ref. 28; TFC reported as mean ± SEM, TFC should be no more than 2% (Affymetrix).

Figure 1

Hierarchical clustering of proximal colon gene expression profiles

Hierarchical 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 individual 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).

Figure 2

Hierarchical clustering of 211 differentially expressed genes in either SPI or WPH

Hierarchical 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 relatedness of different profiles.

Differentially expressed genes

Multiple filtering criteria were applied to the microarray data set so as to identify differentially expressed colon transcripts in rats fed SPI, WPH or CAS; results are reported only for transcripts that passed all three analytical filters used: DMT t-test, SAM and pair-wise comparison survival methods. Among the 8799 genes and ESTs examined with the rat U34A array, we identified 31 induced and 49 repressed genes in proximal colons of SPI-fed rats, whereas 44 induced and 119 repressed genes were detected in WPH-fed rats (Tables 2, 3, 4, 5). Interestingly, more down- than up-regulated genes were noted for both SPI and WPH. Additionally, 37 genes were co-repressed, whereas only two were co-induced by SPI and WPH (Table 6). More than 90% of identified genes in WPH and SPI animals showed the same direction of change relative to CAS. This is visually apparent in the hierarchical clustering output (Fig. 2).

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 adhesion

Embigin

AJ009698

-6.57

Cadherin 17

L46874

-4.8

0.036

Cadherin

X78997

-3.36

0.004

Protein tyrosine phosphatase

M60103

-2.64

0.004

Cytokeratin-8

S76054

-2.71

Trans-Golgi network integral membrane protein TGN38

X53565

-4.92

0.012

Tumor-associated calcium signal transducer 1

AJ001044

-9.37

0.001

Claudin-3

AJ011656

-7.55

0.02

Claudin-9

AJ011811

-5.12

Cell cycle/growth control

Mapk6

M64301

-2.61

0.003

Epithelial membrane protein 1

Z54212

-4.67

0.015

Glucagon

K02813

-7.73

0.005

Peptide tyrosine-tyrosine (YY)

M17523

-4.56

0.001

Src related tyrosine kinase

U09583

-3.31

0.033

FGF receptor activating protein

U57715

-4.25

0.002

Cyclin D1

D14014

-1.97

0.001

Neu oncogene

X03362

-2.61

0.017

Defense/immunity protein

Seminal vesicle secretion protein iv

J00791

-5.35

0.001

Putative cell surface antigen

U89744

-5.22

0.008

Decay accelerating factor GPI

AF039583

-6.12

Beta defensin-1

AF093536

-26.78

0.001

Detoxification/antioxidation

Glutathione S-transferase

J02810

-5.17

Glutathione S-transferase Yb

X04229

-9.33

J03914

-2.43

0.002

Glutathione S-transferase, alpha 1

K01932

-3.07

0.002

Glutathione transferase, subunit 8

X62660

-6.42

0.001

Glutathione S-transferase Yc1

S72505

-3.69

0.004

Glutathione S-transferase Yc2

S72506

-21.38

0.008

N-acetyltransferase 1

U01348

-4.64

0.003

Cytochrome P450CMF1b

J02869

-8.23

0.001

Cytochrome P450 4F4

U39206

-6.43

0.004

Cytochrome P450 monooxygenase

U39943

-2.82

0.011

Cytochrome P450 pseudogene

U40004

-2.87

Cytochrome P450 3A9

U46118

-6.91

Cytochrome P450IVF

M94548

-5.78

0.002

Cytochrome P450, subfamily 51

U17697

-2.07

0.005

Alcohol dehydrogenase

M15327

-2.06

0.025

Aldehyde dehydrogenase

M23995

-10.56

0.035

AF001898

-2.72

0.004

D-amino-acid oxidase

AB003400

-13.69

3-methylcholanthrene-inducible UDP-glucuronosyltransferase

S56937

-9

UDP-glucuronosyltransferase

D38062

-3.17

0.005

D38065

-3.29

0.002

UDP glycosyltransferase 1

D83796

-6.87

J02612

-6.58

J05132

-4.03

Metabolism

Meprin 1 alpha

S43408

-3.82

0.014

Brain serine protease bsp1

AJ005641

-4.42

0.007

Cystathionine gamma-lyase

D17370

-3.05

0.002

Cathepsin S

L03201

-2.62

Meprin beta-subunit

M88601

-5

0.004

Disintegrin and metalloprotease domain 7

X66140

-11.91

Fucosyltransferase 1

AB006137

-4.96

0.001

Fucosyltransferase 2

AB006138

-7.97

0.017

UDP-glucose:ceramide glycosyltransferase

AF047707

-2.86

0.007

Type II Hexokinase

D26393

-2.7

0.001

Hexokinase II

S56464

-4.45

0.007

CDP-diacylglycerol synthase

AB009999

-4.66

Carboxylesterase precursor

AB010635

-5.29

0.002

Fatty acid Coenzyme A ligase

AB012933

-2.5

0.041

3beta-HSD

L17138

-3.27

11-beta-hydroxylsteroid dehydrogenase type 2

U22424

-3

0.001

Peroxiredoxin 6

AF014009

-3.55

0.01

Platelet phospholipase A2

X51529

-3.25

0.001

Ligand binding/carrier

Carnitine transporter

AB017260

-3.95

0.005

Chloride channel (ClC-2)

AF005720

-5.69

0.002

Putative potassium channel

AF022819

-4.84

Mitochondrial dicarboxylate carrier

AJ223355

-3.55

0.009

Aquaporin 3

D17695

-7.83

Na_H_Exchanger

L11236

-9.81

0.003

Angiotensin/vasopressin receptor (AII/AVP)

M85183

-3.3

0.002

H+, K+-ATPase

M90398

-13.87

Intestinal fatty acid binding protein

K01180

-7.29

0.001

Apolipoprotein A-I precursor

M00001

-3.45

0.023

Apolipoprotein A-I

J02597

-2.47

0.003

Sodium-hydrogen exchange protein-isoform 3

M85300

-7.36

0.004

Liver fatty acid binding protein

V01235

-2.62

Sodium transporter

X59677

-3.8

Cation transporter

X78855

-3.62

0.003

ATP-binding cassette

AB010467

-3.89

0.004

Methionine adenosyltransferase II, alpha

J05571

-2.91

0.007

Phenylalanine hydroxylase

M12337

-7.43

Carbonic anhydrase IV

S68245

-4.28

0.011

Signal transduction

B7 antigen

X76697

-170.95

0.002

CD24 antigen

U49062

-3.08

Chemokine CX3C

AF030358

-5.04

0.011

Itmap1

AF022147

-7.5

0.001

HCNP

E05646

-2.5

0.001

Brain glucose-transporter protein

M13979

-2.97

0.019

Protein kinase C delta

M18330

-2.48

0.002

Guanylate cyclase 2C

M55636

-4.58

0.003

A2b-adenosine receptor

M91466

-2.8

0.04

Guanylate cyclase activator 2A

M95493

-4.18

0.005

Phospholipase C beta-3

M99567

-2.57

0.018

Tm4sf3

Y13275

-3.33

Phospholipase D

AB000778

-2.71

0.009

BEM-2

D45413

-6.41

0.015

Sgk

L01624

-3.93

Stress response/apoptosis

Prostaglandin D synthetase

J04488

-43.11

0.009

GTP cyclohydrolase I

M58364

-3.26

0.014

Structure proteins

Chromogranin B (Chgb)

AF019974

-2.56

0.005

Intestinal mucin

M76740

-5.09

0.002

Muc3

U76551

-11.07

0.006

Mucin-like protein

M81920

-11.97

0.001

Myosin 5B

U60416

-3.94

Keratin 18

X81448

-3.23

0.004

Keratin 19

X81449

-2.69

0.001

ZG-16p protein

Z30584

-4.43

0.002

Plasmolipin

Z49858

-7.2

Cytokeratin 21

M63665

-4.96

Syndecan

S61865

-3.3

0.006

Claudin 3

M74067

-6.68

0.01

Transcription factor/regulator

Hepatocyte nuclear factor 3 gamma

AB017044

-6.96

Apolipoprotein B mRNA editing protein

L07114

-2.34

DNA-binding inhibitor

L23148

-4.1

0.01

Kruppel-like factor 4 (gut)

L26292

-3.08

0.017

Others

Prolactin receptor

M74152

-3.26

0.014

LOC286964

U89280

-2.96

0.003

Ckmt1

X59737mRNA

-2.65

0.025

Arginase II

U90887

-23.69

Deleted in malignant brain tumors 1

U32681

-3.47

0.002

3' end GAA-triplet repeat

L13025

-2.73

0.001

Polymeric immunoglobulin receptor

L13235

-2.93

0.004

*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), 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 adhesion

Fibronectin

X05834

2.3

EGF-containing fibulin-like extracellular matrix protein 1

D89730

2.17

0.004

Cell cycle/growth control

Somatostatin

M25890

2.72

0.001

Somatostatin-14

K02248

3.87

0.009

APEG-1

U57097

3.24

0.002

Defense/immunity protein

IgG gamma heavy chain

M28670

2.21

0.009

T-cell receptor beta chain

X14319

2.14

Adipsin

M92059

3.21

Ligand binding/carrier

Angiotensin receptor

M86912

2.75

0.017

Calretinin

X66974

2.52

0.005

Purkinje cell protein 4

M24852

3.06

0.001

Secretogranin III

U02983

2.77

0.005

Secretogranin II

M93669

2.84

0.001

Aquaporin 1

X67948

3.4

0.008

Cacna2d1

M86621

2.84

Retinol-binding protein

M10934

2.17

0.018

Metabolism

Lipoprotein lipase

L03294

2.72

Ubiquitin carboxyl-terminal hydrolase

D10699

0.003

Signal transduction

Thy-1 protein

X02002

2.89

0.002

CD3 gamma-chain

S79711

3.28

0.002

Synapsin

M27925

3.94

0.001

Alpha-actinin-2 associated LIM protein

AF002281

2.74

0.009

RESP18

L25633

2.74

0.033

T3 delta protein

X53430

2.75

0.003

Protein phosphatase inhibitor-1

J05592t

2.6

0.009

CART protein

U10071

2.16

0.001

Neuroendrocrine protein

M63901

3.7

0.006

Protein kinase C-binding protein Zeta1

U63740

3.14

0.003

cannabinoid receptor 1

X55812

2.17

0.002

Guanylyl cyclase A

J05677

3.18

0.007

Tachykinin 1

X56306

2.36

0.036

Protein tyrosine phosphatase

L19180

2.47

0.041

Argininosuccinate synthetase

X12459

4.69

0.004

Stress response/apoptosis

Small inducible cytokine

Y08358

3.35

0.029

Structure proteins

Fast myosin alkali light chain

L00088

4.52

0.03

Light molecular-weight neurofilament

AF031880

2.41

Neurofilament protein middle

Z12152

2.97

0.006

Alpha-tubulin

V01227

2.25

Peripherin

AF031878

2.82

0.007

Transcription factor/regulator

snRNP

M29293

2.11

0.004

snRNP-associated polypeptide

X73411

3.33

0.002

Others

C1-13 gene product

X52817

3.17

ND5, ND6

S46798

2.31

0.015

Sensory neuron synuclein

X86789

2.84

*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), 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*

Category and Gene Name

Probe Set GB Accession No.

Fold Change

P value

Cell adhesion

Embigin

AJ009698

-5.13

0.001

Cell Cycle/growth control

FGF receptor activating protein 1

U57715

-5.59

0.002

BEST5 protein

Y07704

-2.37

0.003

Peptide tyrosine-tyrosine (YY)

M17523

-3.91

0.002

Glucagon gene

K02813

-6.58

0.002

Epithelial membrane protein-1

Z54212

-3.47

0.017

Neu oncogene

X03362

-1.58

0.05

Defense/immunity protein

Beta defensin-1

AF068860

-42.16

0.001

AF093536

-10.2

Detoxification/antioxidation

Glutathione S-transferase

J02810

-7.14

Glutathione S-transferase Yb

X04229

-11.71

0.001

Glutathione S-transferase, alpha 1

K01932

-4.18

0.004

Glutathione S-transferase Yc1

S72505

-5.23

0.001

Glutathione S-transferase Yc2

S72506

-5.27

0.012

S82820

-3.45

0.006

Cytochrome P450 4F4 (CYP4F4)

U39206

-6.52

0.002

Cytochrome P450CMF1b

J02869

-4.12

0.002

Cytochrome P450 (CYP4F1)

M94548

-2.88

0.002

1-Cys peroxiredoxin

Y17295

-2.55

0.002

Metallothionein

M11794

-2.92

0.006

D-amino-acid oxidase

AB003400

-5.42

Peroxiredoxin 6

AF014009

-3.07

0.008

Phenylalanine hydroxylase

M12337

-10.99

0.001

Metabolism

Dipeptidase

L07315

-3.08

0.001

Meprin beta-subunit

M88601

-3.27

0.001

Disintegrin and metalloprotease domain 7

X66140

-14.03

Ligand binding/carrier

Carnitine transporter

AB017260

-3.81

0.003

Chloride channel (ClC-2)

AF005720

-3.26

0.001

Putative potassium channel

AF022819

-2.69

0.001

Mitochondrial dicarboxylate carrier

AJ223355

-2.54

0.01

Aquaporin 3

D17695

-4.13

Intestinal fatty acid binding protein

K01180

-4.43

0.005

Na_H_Exchanger

L11236

-4.47

0.002

H+, K+-ATPase

M90398

-2.52

0.001

Carbonic anhydrase IV

S68245

-4.28

0.005

Sodium transporter

X59677

-3.4

Phosphatidylethanolamine binding protein

X75253

-2.69

Signal transduction

B7 antigen

X76697

-170.95

0.002

HCNP

E05646

-3.38

Itmap1

AF022147

-7.97

0.005

Guanylate cyclase activator 2A

M95493

-3.28

0.006

Sgk

L01624

-2.76

Stress response/apoptosis

Prostaglandin D synthetase

J04488

-45.8

0.01

Structure proteins

Muc3

U76551

-3.56

0.01

Intestinal mucin

M76740

-3.31

0.006

Mucin-like protein

M81920

-3

Plasmolipin

Z49858

-2.92

0.003

Transcription factor/regulator

Testis specific X-linked gene

X99797

-6.91

0.003

Others

Arginase II

U90887

-3.22

3-phosphoglycerate dehydrogenase

X97772

-4.15

0.017

Aldehyde dehydrogenase family 1

AF001898

-3.93

0.004

*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), 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 5

Up-regulated genes in rats fed with SPI diet*

Category and Gene Name

Probe Set GB Accession No.

Fold Change

P value

Cell adhesion

Collagen alpha1 type I

Z78279

2.49

Secreted phosphoprotein 1

M14656

111.39

0.006

Matrix metalloproteinase 13

M60616

24.34

0.002

Regenerating islet

M62930

193.08

0.011

Defense/immunity protein

Ig gamma-2a chain

L22654

115.17

0.001

Ig gamma heavy chain

M28670

3.22

Ig germline kappa-chain C-region

M18528

2.48

0.038

Ig light-chain

U39609

2.63

0.021

Fc-gamma

M32062

4.72

0.017

Detoxification

Glutathione S-transferase 1

J03752

2.86

Glutathione-S-transferase,alpha type2

K00136

2.56

0.009

UDP glucuronosyltransferase

D38066

2.83

0.014

Metabolism

Matrix metalloproteinase 7

L24374

3.63

0.02

lysozyme

rc_AA892775

2.77

Matrix metalloproteinase 12

X98517

11.8

0.013

Mitochondrial carbamyl phosphate synthetase I

M12335

59.25

0.001

Aldolase B, exon 9

X02291

8.7

0.01

Aldolase B, exon 2

X02284

2.71

0.001

Signal transduction

MHC class II antigen RT1.B-1 beta-chain

X56596

2.55

0.001

CD3 gamma-chain

S79711

4.51

0.001

Ligand binding/carrier

Intracellular calcium-binding protein

L18948

28.29

0.014

Retinol binding protein II

M13949

5.11

0.001

Apolipoprotein B

M27440

6.47

0.024

Apolipoprotein A-I

J02597

2.49

0.004

Iron ion transporter

AF008439

18.78

0.008

Stress response/apoptosis

Heme oxygenase

J02722

9.66

0.002

JE product

X17053

3.52

0.001

Pancreatitis-associated protein

M98049

68.39

0.004

Pancreatitis associated protein III

L20869

15.35

Reg protein

E01983

30.25

0.001

Others

Histamine N-tele-methyltransferase

S82579

6.17

0.04

*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), 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.

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 genes

Embigin

AJ009698

-6.57

-5.13

0.001

Epithelial membrane protein 1

Z54212

-4.67

0.015

-3.47

0.017

Glucagon

K02813

-7.73

0.005

-6.58

0.002

Peptide tyrosine-tyrosine (YY)

M17523

-4.56

0.001

-3.91

0.002

FGF receptor activating protein

U57715

-4.25

0.002

-5.59

0.002

Neu oncogene

X03362

-2.61

0.017

-1.58

0.05

CD52 antigen

X76697

-170.95

0.002

-170.95

0.002

Beta defensin-1

AF068860

-54.48

0.001

-42.16

0.001

Glutathione S-transferase

J02810

-5.17

-7.14

Glutathione S-transferase Yb

X04229

-9.33

-11.71

0.001

Glutathione S-transferase, alpha 1

K01932

-3.07

0.002

-4.18

0.004

Glutathione S-transferase Yc1

S72505

-3.69

0.004

-5.23

0.001

Glutathione S-transferase Yc2

S72506

-21.38

0.008

-5.27

0.012

Cytochrome P450CMF1b

J02869

-8.23

0.001

-4.12

0.002

Cytochrome P450 4F4

U39206

-6.43

0.004

-6.52

0.002

Cytochrome P450IVF

M94548

-5.78

0.002

-2.88

0.002

D-amino-acid oxidase

AB003400

-13.69

-5.42

Meprin beta-subunit

M88601

-5

0.004

-3.27

0.001

Disintegrin and metalloprotease domain 7

X66140

-11.91

-14.03

Carnitine transporter

AB017260

-3.95

0.005

-3.81

0.003

Chloride channel (ClC-2)

AF005720

-5.69

0.002

-3.26

0.001

Putative potassium channel

AF022819

-4.84

-2.69

0.001

Mitochondrial dicarboxylate carrier

AJ223355

-3.55

0.009

-2.54

0.01

Aquaporin 3

D17695

-7.83

-4.13

Na_H_Exchanger

L11236

-9.81

0.003

-4.47

0.002

H+, K+-ATPase

M90398

-13.87

-2.52

0.001

Fatty acid binding protein 1

K01180

-7.29

0.001

-4.43

0.005

Sodium transporter

X59677

-3.8

-3.4

Carbonic anhydrase IV

S68245

-4.28

0.011

-4.28

0.005

Itmap1

AF022147

-7.5

0.001

-7.97

0.005

HCNP

E05646

-2.5

0.001

-3.38

Guanylate cyclase activator 2A

M95493

-4.18

0.005

-3.28

0.006

Sgk

L01624

-3.93

-2.76

Prostaglandin D synthetase

J04488

-43.11

0.009

-45.8

0.01

Mucin 3

M76740

-5.09

0.002

-3.31

0.006

Mucin-like protein

M81920

-11.97

0.001

-3

Plasmolipin

Z49858

-7.2

-2.92

0.003

Up-regulated genes

Ig gamma heavy chain

M28670

2.21

0.009

3.22

CD3 gamma-chain

S79711

3.28

0.002

4.51

0.001

*Changes in gene expression were determined by t-test (DMT), comparative analysis (MAS 5.0), 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.

Gene expression: effects of WPH

As based on Gene Ontology (GO) annotations, the 44 up-regulated and 119 down-regulated genes of the WPH group belong to multiple functional categories including cell 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), and others (Tables 2 &3). The fold change for up-regulated genes ranged between 2.1 [small nuclear ribonucleoparticle-associated protein (snRNP)] to 4.7 (argininosuccinate synthetase), whereas down-regulated genes exhibited fold changes between 2.0 (cyclin D1) and 171 (CD52 antigen).

Lifetime ingestion of WPH affected the expression of xenobiotic metabolism-related enzymes including several of the cytochrome P450s and glutathione S-transferases, alcohol dehydrogenase (ADH), and UDP-glucuronosyltransferase. Cytochrome P450 enzymes and ADH are considered to play key roles in activation of the proximate carcinogen from AOM 29. Down-regulation of expression of Phase I detoxification enzymes by WPH might therefore diminish AOM-induced DNA adducts and genomic instability. Consistent with results from a study in which whey proteins inhibited cell proliferation in vitro 11, lifetime feeding of WPH was associated with changes in expression of genes involved in cell cycle control and proliferation; cyclin D1, neu oncogene, mapk6, glucagon, and peptide YY (PYY) genes were down-regulated, whereas the expression of somatostatin, a growth-inhibitory peptide was induced. WPH altered expression of genes involved in cellular defense. Induced genes included Ig gamma heavy chain, adipsin, and T-cell receptor beta chain, whereas expression of the antibacterial peptide beta defensin-1 and seminal vesicle secretion protein IV (SVS IV) were down-regulated. About 20% of WPH-affected genes are involved in cell signaling; these include guanylate cyclase 2C, protein kinase C delta, and synapsin. Additionally, genes encoding ligands or membrane channels [i.e., chloride channel, intestinal fatty acid binding protein (I-FABP), apoliprotein A-I (Apo-AI), Na+, K+-ATPase, and sodium transporter] were down-regulated by WPH, whereas calretinin and retinol binding protein (RBP) levels were increased.

Gene expression: effects of SPI

Colon genes, whose mRNA expression was affected by ingestion of SPI, fell into multiple functional categories including cell adhesion (n = 4), cell cycle and growth control (n = 6), detoxification (n = 18), defense and immunity (n = 6), signal transduction (n = 4), transcriptional regulation (n = 1), metabolism (n = 8), ligands and carrier proteins (n = 17), cell death proteins (n = 5), and structural proteins (n = 3) (Tables 4 &5).

Relative abundance of numerous transcripts was changed in the same direction by WPH and SPI (Fig. 2). However, some exceptions were noted. For example, mRNA encoding Apo-AI was down-regulated by WPH, but elevated by SPI. Apo-AI is the major determinant of the capacity of HDL particles to promote cholesterol efflux and this protein 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.

Confirmation of differential gene expression

We performed quantitative real-time RT-PCR on selected genes to confirm the microarray results. Based upon known associations with cell proliferation or differentiation, 14 genes were chosen for further study. Included in this group was BTEB2; this gene was not present on the microarrays but was included in RT-PCR analysis due to its significant expression in intestine and involvement in cell proliferation [see discussion]. As shown in Figure 3, eight genes were confirmed to be differentially expressed: these included the gastrointestinal hormone genes PYY (12.9-fold down-regulated in WPH fed rats; P = 0.004), glucagon (17.8-fold down-regulated in WPH fed rats; P = 0.005), and somatostatin (3.92-, 2.65-fold up-regulated in SPI and WPH fed rats; P = 0.05, P = 0.025, respectively); cyclin D1 (1.6-, 2.4-fold down-regulated in SPI and WPH fed rats; P = 0.033, P = 0.001, respectively); BTEB2 (1.9-, 6.7-fold down-regulated in SPI and WPH fed rats; P = 0.024, P < 0.001, respectively); c-neu proto-oncogene (2.5-, 4.1-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively); the colonocyte differentiation marker I-FABP (2.9-, 4.0-fold down-regulated in SPI and WPH fed rats; P = 0.023, P = 0.01, respectively); and the mucin, MUC3 (2.78-, 4.05-fold down-regulated in SPI and WPH fed rats; P < 0.001, P < 0.001, respectively). Differential expression of five other genes was not confirmed statistically, due to individual animal variation in the transcript levels; however, the mean-fold changes for mRNA abundance were greater than two and in agreement with the corresponding microarray results for these genes. Only one of the selected genes – retinol binding protein (RBP), failed to exhibit greater than a 2-fold change (in the predicted direction) at the mRNA level by real-time RT-PCR.

Figure 3

Quantitative real-time RT-PCR verification of microarray results

Quantitative 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 ± SEM and were analyzed by one-way ANOVA, *P < 0.05, SPI or WPH vs. CAS.

Serum somatostatin (Sst)

As shown in Fig. 4, circulating Sst concentration was significantly higher in rats fed WPH and SPI. Colonic Sst protein content in colon homogenates was below the limit of detection of the assay used (data not shown).

Figure 4

Diet effects on serum Sst concentration

Diet effects on serum Sst concentration. Values are mean ± SEM. One-way ANOVA. *P < 0.05, SPI or WPH vs. CAS.

Discussion

The type of dietary protein(s) can markedly affect the onset and/or progression of CRC 31. Epidemiological and animal studies have found that dietary soy and whey proteins decrease the incidence of certain tumors, including those of the colon and rectum 6732333435. Using the AOM-treated male Sprague Dawley rat model, we previously found that lifetime feeding of SPI led to a ~ 76% lower incidence of AOM-induced colon tumors compared to rats lifetime-fed CAS 8. Additionally, in the same studies, a ~ 46% lower incidence of colon tumors was found in WPH-fed compared to CAS-fed rats 9. The molecular mechanism(s) by which these dietary proteins reduce the incidence of chemically-induced colon tumors is unclear, although several mechanisms and putative bio-active factors have been proposed 1112131415161718192021222324. The present study has now identified genes that are differentially expressed as a function of these diets and which serve to highlight potential pathways for dietary protection from carcinogenesis.

The ability to simultaneously analyze a large number of different mRNAs makes microarrays very appealing for identifying genes and gene families whose expression is altered by diet 3637. We focused on the 'normal' colon tissue since we are interested in genes that are differentially regulated by diet and which act in anti-oncogenic fashion in pre-cancerous tissues. We limited our analysis to the proximal colon since several studies have suggested that the molecular etiology of proximal and distal colon tumors differs 2526 and proximal colon tumors have become more prevalent with Westernization of the diet and aging of the population 27. We chose to include colonic smooth muscle with the mucosa since: a) the former tissue layer interacts with the latter to influence its growth and function, and b) we could monitor all colonic genes affected by diet. However, one potential caveat to this strategy is the 'dilution' effect that may have been imposed on the more rare mucosal transcripts. Another caveat is that no information is obtained regarding where the differentially expressed transcripts occur. In this regard, however, we have confirmed by immuno-histochemistry that I-FABP is expressed predominantly in the inter-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 experimental reagents with the minimum number required for statistical analysis. The quantitative PCR analyses provided confirmation that the filtering strategies used yielded bona-fide differentially expressed transcripts.

Figure 5

Immuno-histochemistry for I-FABP in colons from AOM-treated rats

Immuno-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 epithelium (overall intensity of staining is greater for CAS than for WPH).

Only two transcripts were induced by both SPI and WPH; whereas 37 transcripts were repressed by both SPI and WPH. This suggests that the cancer-protective actions of the two diets are generally associated with repression of colonic genes that facilitate tumorigenesis. An alternative explanation is that CAS induces genes that facilitate colon cancer development when compared to SPI and WPH. It is also likely that SPI and WPH diets act in unique ways to inhibit tumorigenesis. Irregardless, our results indicate that the nature of the dietary protein can profoundly affect colon gene expression profiles. Thus, gene expression profiling studies of colons should account for potential confounding effects of diet.

Dietary factors in SPI or WPH inhibit cell proliferation and induce apoptosis among other biological actions 1113. In the present study, we identified cyclin D1 gene and the neu proto-oncogene as being repressed in proximal colon by SPI and WPH. Cyclin D1 is a key regulator of cell cycle progression 38, and a target of β-catenin, a protein whose abnormal accumulation in the nucleus is strongly linked to the development of multiple tumor types, including those of the colon 39. Aberrantly increased expression of cyclin D1 in colon epithelial cells contributes to their abnormal proliferation and tumorigenicity 4041. Similarly, the oncogenic and cellular growth-promoting activities of the HER-2/neu proto-oncogene are well known 42. HER-2/neu, a tyrosine kinase receptor for neu-differentiation factor, is expressed in normal colonic epithelium and is up-regulated in adenomatous polyps of the colon 43. The down regulation of cyclin D1 and c-neu mRNA abundance by SPI and WPH may at least partly explain their anti-tumorigenic properties. Similarly, Krüppel-like transcription factors have been linked to cell growth and tumorigenesis. BTEB2 (also known as Krüppel-like factor 5, KLF5, or intestinal KLF) was reported to enhance intestinal epithelia cell colony formation, cyclin D1 transcription, and cell proliferation 44. Consistent with our results for cyclin D1, colonic BTEB2 mRNA expression was down-regulated by SPI and WPH. Aquaporin 3, a water channel highly expressed in colonic epithelium, was down-regulated by SPI and WPH. Aquaporins are thought to be induced early in colon cancer and to facilitate oncogenesis 45, therefore, dietary repression of such genes may additionally contribute to anti-tumorigenesis. The results for I-FABP and MUC3 indicated 3–4 fold decreases in transcript abundance in proximal colons of rats on SPI or WPH diets. These particular results are not easily reconciled with decreased tumorigenesis in SPI and WPH groups, since both genes are highly expressed in the normal differentiated colonic epithelium and are likely to be under-expressed in adenomas and adenocarcinomas 46. Perhaps, these represent diet-modulated genes that are not direct participants in anti-tumorigenesis.

Gastrointestinal hormones regulate a myriad of intestinal functions including motility, absorption, digestion, cell proliferation and death, and immune response 47. The microarray and real-time RT-PCR assays identified inductive effects of SPI and WPH on somatostatin mRNA and protein abundance. These results implicate this gene product in autocrine and paracrine mechanisms underlying colon cancer protection by SPI and WPH since somatostatin is a well-known anti-proliferative agent for colon tumor cells 4849. This hormone is also a negative regulator of angiogenesis 50; this is predicted to counter tumorigenesis. It is possible that the small decrements in rat growth rates observed with lifetime SPI or WPH diets 89 are a consequence of this increased circulating somatostatin level. We also found decreased abundance of mRNAs encoding peptide YY (PYY) and glucagon in colons of WPH-fed rats. PYY gene expression in human colon tumors is much reduced relative to the adjacent normal tissue 51; however, chemically-induced colon tumors in rats generally exhibit higher overall expression of PYY due to increased prevalence of PYY-positive cells, compared to normal mucosa 5253. PYY stimulates proliferation of intestinal epithelium 54; therefore, an inhibition of PYY expression by dietary WPH may contribute to colon cancer-protective actions. A similar scenario might apply to colon glucagon gene expression, as this growth stimulatory peptide for colon cancer cells 55 was inhibited by WPH at the level of colon mRNA abundance.

Our data highlighted other aspects of diet and colon gene expression that warrant further study. For example, the B7 antigen (also known as CD52) mRNA was strongly down-regulated by SPI or WPH. The corresponding protein is normally expressed at high levels on cell membranes of T and 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-tumor colon tissue of rats on SPI or WPH diets may reflect fewer numbers of immune cells in this tissue, as compared to CAS-fed animals. One possible interpretation of this data is that the 'normal' tissue of the CAS group has manifested an immune response, perhaps in response to increased tumorigenicity relative to SPI or WPH groups. Such an interpretation raises the prospect of a functional immuno-editing mechanism 57 occurring in this model of colon cancer and an indirect effect of diet on lymphocyte populations (via presence of tumors or tumor precursors) in the colon. An alternative mechanism is that dietary protein can directly affect the populations of lymphocytes resident in the colon, which in turn, may affect tumorigenesis. A related observation was the enhanced abundance of CD3 gamma chain transcripts in colons of SPI and WPH animals. The protein encoded by this transcript helps mediate T cell antigen receptor engagement and signaling 58; its decreased abundance in colonic T cells of CAS-fed animals may indicate a specific immune defect 59 occurring in the CAS-fed animals after exposure to carcinogen and thereby contributing to enhanced tumorigenesis in this group.

Several microarray studies of human paired normal colon vs. colon tumors have been published 6061626364. Comparison of the present results for normal colon tissue of AOM-treated rats on different diets to the published studies for human CRC identified only a small number of common differentially expressed genes and/or gene families in common (data not shown). This small number is probably due to the fact that our study did not examine colon tumors; rather we focused on 'normal' colon tissue. In this regard, it will be interesting to examine the expression profiles of colons from animals not treated with AOM so as to more specifically correlate transcripts with diet and cancer phenotype. This study has illuminated a number of genes and gene families that may act as dietary protein-dependent modulators of oncogenesis in the rat colon. Additional studies that specifically address the functional involvement of these genes in cancer-prevention via dietary means are required to confirm the postulated roles.

Conclusions

We have identified genes in rat colon that are differentially expressed, as a consequence of altered dietary protein, during AOM-induced oncogenesis. These are candidates for genes that sub-serve the anti-cancer effects of dietary SPI and WPH in this tissue.

Methods

Rats, diets and carcinogen treatment

The animals whose colons were used in the present study have been previously described 89. Time-mated [gestation day (GD) 4] Sprague-Dawley rats were purchased from Harlan Industries (Indianapolis, IN), housed individually and allowed ad libitum access to water and pelleted food. Rats were randomly assigned to one of three semi-purified isocaloric diets made according to the AIN-93G diet formula 65 and which differed only by protein source: a) CAS (New Zealand Milk Products, Santa Rosa, CA), b) WPH (New Zealand Milk Products, Santa Rosa, CA) or c) SPI (Dupont Protein Technologies, St. Louis, MO). Offspring were weaned to the same diet as their mothers and were fed the same diets throughout the study. At 90 days of age, male offspring received s.c. injections of 15 mg/kg AOM (Ash Stevens, Detroit, MI) in saline once a week for 2 weeks. Forty weeks later, rats were euthanized, and the colon (cecum to anus) was divided into two equal segments (proximal and distal), opened longitudinally, and examined for tumors. We found that both WPH and SPI significantly decreased the colon tumor incidence [data published in 89]. A representative non-tumor segment of each proximal colon (PC) was frozen in liquid nitrogen and stored at -80°C for later use. Animal care and handling were in accordance with the Institutional Animal Care & Use Committee guidelines of the University of Arkansas for Medical Sciences.

RNA processing

Total RNA was isolated from rat proximal colons (n = 7 for each of CAS, SPI and WPH diets) using TRIzol reagent (Invitrogen, Carlsbad, CA), and further purified with the RNeasy Mini Kit (QIAGEN, Valencia, CA). To remove contaminating DNA, on-column DNA digestion with RNase-Free DNase (QIAGEN) was performed. Integrity of isolated RNAs was confirmed using the RNA 6000 Nano LabChip kit with the Agilent 2100 Bioanalyzer System (Agilent Biotechnologies, Palo Alto, CA). To reduce errors due to biological variability, RNA samples were pooled as proposed by Bakay et al 66. Pooled RNA (equal amounts of RNA from each of 7 animals; 8 ug total) was used for cDNA synthesis using a T7-(deoxythymidine)₂₄primer and Superscript II (Life Technologies, Inc., Gaithersburg, MD). The resulting cDNA was used with the ENZO BioArray High Yield RNA Transcript labeling kit (ENZO, Farmingdale, NY) to synthesize biotin-labeled cRNA. The cRNA was purified on RNeasy spin columns (QIAGEN) and subjected to chemical fragmentation (size range of 35 to 200 bp). Three replicate cRNA targets were made in parallel starting from each RNA pool.

Microarray procedures

Ten ug of cRNA was hybridized for 16 hours to an Affymetrix (Santa Clara, CA) rat U34A GeneChip (3 chips used per diet group), followed by incubations with streptavidin-conjugated phycoerythrin, and then with polyclonal anti-streptavidin antibody coupled to phycoerythrin. Following washing, GeneChips were scanned using an Agilent GeneArray laser scanner. Images were analyzed using Affymetrix MAS 5.0 software. Bacterial sequence-derived probes on the arrays served as external controls for hybridization, whereas the housekeeping genes β-actin and GAPDH served as endogenous controls and for monitoring the quality of the RNA target. To compare array data between GeneChips, we scaled the average of the fluorescent intensities of all probes on each array to a constant target intensity of 500.

Bioinformatics

To validate the microarray procedure for our samples, unsupervised nearest-neighbor hierarchical clustering (Spotfire, Somerville, MA) was performed on gene expression data. The inter-chip variability test also was performed as specified in the Affymetrix data analysis manual 28. To identify colon genes differentially expressed with SPI or WPH (control: CAS diet), multiple criteria were applied; final results are reported only for transcripts that passed all three analytical steps described below. Firstly, the t-test feature of DMT (Affymetrix) was used to identify genes whose expression was regulated (induced/repressed with P < 0.05) by SPI or WPH, and signal fold changes (FC) for these genes were calculated. Secondly, microarray data were analyzed using 'Significance of Analysis of Microarrays' (SAM, Stanford) to identify significant changes in gene expression among diet groups 67, using a false discovery rate (FDR) cutoff of 0.5%. Lastly, a pair-wise comparison survival (3 × 3) method was used to identify differentially expressed transcripts 68. In brief, the three replicate expression profiles obtained for SPI colons were iteratively compared with the three CAS profiles (latter as baseline) in MAS 5.0 (Affymetrix), generating nine comparisons in total. Transcripts with a log ratio greater than or equal to 1 (≥2 fold change), which increased (I) in nine of nine comparisons, and which were expressed above background (i.e., called as Present) in all three SPI GeneChips, were considered to be up-regulated by SPI. Transcripts with a log ratio less than or equal to -1, were decreased (D) in nine of nine comparisons, and expressed above background (Present) in all three CAS chips were considered to be down-regulated by SPI. WPH-regulated genes were similarly identified. Genes that were independently identified by all three approaches comprised the final reported lists of differentially expressed genes (Tables 2, 3, 4, 5, 6).

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

One μg of total RNA from each of the 21 individual proximal colons (which comprised the original pools for the microarray experiment) was reverse-transcribed using random hexamers and MultiScribe Reverse Transcriptase in a two-step RT-PCR reaction (Applied Biosystems, Foster City, CA). Primers (Table 7) were designed using 'Primer Express' (Applied Biosystems) and were selected to yield a single amplicon; this was verified by dissociation curves and/or analysis in agarose gels. SYBR Green real-time PCR was performed with an ABI Prism 7000 Sequence Detector. Thermal cycling conditions included pre-incubation at 50°C for 2 min, 95°C for 10 min followed by 40 PCR cycles at 95°C for 15 sec and 60°C for 1 min. The relative transcript levels for each gene were calculated using the relative standard curve method (User Bulletin #2, Applied Biosystems) and normalized to the house-keeping gene β-actin. Data are reported as mean ± SEM of n = 7 animals per dietary group. Significant differences between diet groups were determined by one-way ANOVA (P < 0.05).

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_031144

I-FABP

5'-AGGAAGCTTGGAGCTCATGACA-3'

5'-TCCTTCCTGTGTGATCGTCAGTT-3'

K01180

Neu Oncogene

5'-GTGGTCGTTGGAATCCTAATCAA-3'

5'-CCTTCCTTAGCTCCGTCTCTTTTA-3'

X03362

PYY

5'-AGGAGCTGAGCCGCTACTATGC-3'

5'-TTCTCGCTGTCGTCTGTGAAGA-3'

M17523

Glucagon

5'-TGGTGAAAGGCCGAGGAAG-3'

5'-TGGTGGCAAGGTTATCGAGAA-3'

K02813

Somatostatin

5'-GGAAACAGGAACTGGCCAAGT-3'

5'-TGCAGCTCCAGCCTCATCTC-3'

K02248

PAP III

5'-AAGAGGCCATCAGGACACCTT-3'

5'-CACTCCCATCCACCTCTGTTG-3'

L20869

CYP4F1

5'-CCAAGTGGAAACGGTTGATTTC-3'

5'-TCCTGGCAGTTGCTGTCAAAG-3'

M94548

GST

5'-ACTTCCCCAATCTGCCCTACTTA-3'

5'-CGAATCCGCTCCTCCTCTGT-3'

X04229

Cyclin D1

5'-TCAAGTGTGACCCGGACTGC-3'

5'-ACTTCCCCTTCCTCCTCGGT-3'

D14014

Beta defensin-1

5'-TCTTGGACGCAGAACAGATCAATA-3'

5'-TCCTGCAACAGTTGGGCTATC-3'

AF093536

H+, K+-ATPase

5'-ATTCCGCATCCCTAGACAACG-3'

5'-TCTTACTAAAGCTGGCCATGATGTT-3'

M90398

Prostaglandin D synthetase

5'-CAAGCTGGTTCCGGGAGAAG-3'

5'-TTGGTCTCACACTGGTTTTTCCTTA-3'

J04488

RBP

5'-TCGTTTCTCTGGGCTCTGGTAT- 3'

5'-TTCCCAGTTGCTCAGAAGACG-3'

M10934

Muc3

5'-AAGGTGTGAGGAAGTGATGGAGA-3'

5'-GCAGAGACCGTCGGCTTTATC-3'

U76551

BTEB1

5'-ACACTGGTCACCATCGCCAA-3'

5'-GGACTCGACCCAGATTCGGT-3'

NM_057211

BTEB2

5'-CTACTTTCCCCCATCACCACC-3'

5'-GAATCGCCAGTTTCGAAGCA-3'

AB096709

Serum Sst

Rat serum Sst content (15 animals from each diet) was determined using the somatostatin-28 EIA kit purchased from Phoenix Pharmaceuticals Corporation (Belmont, California).

Authors' contributions

RX performed the microarray and real-time PCR experiments, conducted the data analysis, and participated in drafting the manuscript. TMB designed and oversaw the animal component of the study. FAS designed the analytical and overall approaches to the study, supervised the project, and drafted the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Dr. Rosalia C.M. Simmen and Dr. Rick Helm for insightful comments on the manuscript and Amanda L. Linz for performing the I-FABP immuno-histochemistry. Supported by USDA CRIS 6251-5100-002-06S.

Cancer statistics 2004 – a presentation from the American Cancer Society http://www. cancer.org/downloads/PRO/Cancer%20Statistics%202004.ppt A snapshot of colorectal cancer http://prg.nci.nih.gov/snapshots/Colorectal-Snapshot.pdf Epidemiology and risk factors for colorectal cancer Sandler RS Gastroenterol Clin North Am 1996 25 717 735 8960889 Nutrition and colorectal cancer Potter JD Cancer Causes Control 1996 7 127 146 10.1007/BF00115644 8850441 Food groups and colorectal cancer risk Levi F Pasche C La Vecchia C Lucchini F Franceschi S Br J Cancer 1999 79 1283 1287 10.1038/sj.bjc.6690206 10098773 Most effective colon cancer chemopreventive agents in rats: a systematic review of aberrant crypt foci and tumor data, ranked by potency Corpet DE Tache S Nutr Cancer 2002 43 1 21 10.1207/S15327914NC431_1 12467130 Progress in cancer chemoprevention Kelloff GJ Crowell JA Steele VE Lubet RA Boone CW Malone WA Hawk ET Lieberman R Lawrence JA Kopelovich L Ali I Viner JL Sigman CC Ann N Y Acad Sci 1999 889 1 13 10668477 Soy protein isolate consumption protects against azoxymethane-induced colon tumors in male rats Hakkak R Korourian S Ronis MJ Johnston JM Badger TM Cancer Lett 2001 166 27 32 10.1016/S0304-3835(01)00441-4 11295283 Dietary whey protein protects against azoxymethane-induced colon tumors in male rats Hakkak R Korourian S Ronis MJ Johnston JM Badger TM Cancer Epidemiol Biomarkers Prev 2001 10 555 558 11352868 Whey proteins protect more than red meat against azoxymethane induced ACF in Wistar rats Belobrajdic DP McIntosh GH Owens JA Cancer Lett 2003 198 43 51 10.1016/S0304-3835(03)00307-0 12893429 Growth rates of a human colon adenocarcinoma cell line are regulated by the milk protein alpha-lactalbumin Sternhagen LG Allen JC Adv Exp Med Biol 2001 501 115 120 11787673 Dietary soy isoflavones and estrone protect ovariectomized ERα KO and wild-type mice from carcinogen-induced colon cancer Guo JY Li X Browning JD Jr Rottinghaus GE Lubahn DB Constantinou A Bennink M MacDonald RS J Nutr 2004 134 179 182 14704314 Antiproliferative effects of isoflavones on human cancer cell lines established from the gastrointestinal tract Yanagihara K Ito A Toge T Numoto M Cancer Res 1993 53 5815 5821 8242641 Isoflavones inhibit intestinal epithelial cell proliferation and induce apoptosis in vitro Booth C Hargreaves DF Hadfield JA McGown AT Potten CS Br J Cancer 1999 80 1550 1557 10408396 Dietary flavone is a potent apoptosis inducer in human colon carcinoma cells Wenzel U Kuntz S Brendel MD Daniel H Cancer Res 2000 60 3823 3831 10919656 In vivo administration of genistein has no effect on small intestinal epithelial proliferation and apoptosis, but a modest effect on clonogen survival Booth C Hargreaves DF O'Shea JA Potten CS Cancer Lett 1999 144 169 175 10.1016/S0304-3835(99)00220-7 10529017 Dietary soy sphingolipids suppress tumorigenesis and gene expression in 1,2-dimethylhydrazine-treated CF1 mice and <it>Apc</it><it>Min</it>/+ mice Symolon H Schmelz EM Dillehay DL Merrill AL Jr J Nutr 2004 134 1157 1161 15113963 Soy and whey proteins downregulate DMBA-induced liver and mammary gland CYP1 expression in female rats Rowlands JC He L Hakkak R Ronis MJ Badger TM J Nutr 2001 131 3281 3287 11739881 Soy feeding induces phase II enzymes in rat tissues Appelt LC Reicks MM Nutr Cancer 1997 28 270 275 9343836 Soy induces phase II enzymes but does not inhibit dimethylbenz[<it>a</it>]anthracene-induced carcinogenesis in female rats Appelt LC Reicks MM J Nutr 1999 129 1820 1826 10498753 Dairy proteins protect against dimethylhydrazine-induced intestinal cancers in rats McIntosh GH Regester GO Le Leu RK Royle PJ Smithers GW J Nutr 1995 125 809 816 7722681 Whey proteins in cancer prevention Bounous G Batist G Gold P Cancer Lett 1991 57 91 94 10.1016/0304-3835(91)90200-2 2025891 Whey protein concentrate (WPC) and glutathione modulation in cancer treatment Bounous G Anticancer Res 2000 20 4785 4792 11205219 Colon cancer chemoprevention with ginseng and other botanicals Wargovich MJ J Korean Med Sci 2001 16 Suppl S81 86 11748382 Are right- and left-sided colon neoplasms distinct tumors? Distler P Holt PR Dig Dis 1997 15 302 311 9359018 Anatomical site-specific response to DNA damage is related to later tumor development in the rat azoxymethane colon carcinogenesis model Hong MY Chapkin RS Morris JS Wang N Carroll RJ Turner ND Chang WC Davidson LA Lupton JR Carcinogenesis 2001 22 1831 1835 10.1093/carcin/22.11.1831 11698346 Continuing trends in the prevalence of right-sided lesions among colorectal carcinomas Cady B Stone MD Wayne J Arch Surg 1993 128 505 509 8489383 Affymetrix, GeneChip expression Analysis – Data analysis fundamentals (Manual) 2002 Differential effects of CYP2E1 status on the metabolic activation of the colon carcinogens azoxymethane and methylazoxymethanol Sohn OS Fiala ES Requeijo SP Weisburger JH Gonzalez FJ Cancer Res 2001 61 8435 8440 11731424 Apolipoprotein A-I-containing particles and reverse cholesterol transport: evidence for connection between cholesterol efflux and atherosclerosis risk Fruchart JC De Geteire C Delfly B Castro GR Atherosclerosis 1994 110 Suppl S35 S39 7857382 Soybean phytoestrogen intake and cancer risk Adlercreutz CH Goldin SL Gorbach KA Hockerstedt S Watanabe EK Hamalainen MH Makkanen TH Wahala KT Adlercreutz T J Nutr 1995 125 757S 770S 7884562 Prevention of colon carcinogenesis and carcinoma metastasis by orally administered bovine lactoferrin in animals Tsuda H Sekine K Takasuka N Toriyama-Baba H Iigo M Biofactors 2000 12 83 88 11216510 Dietary milk proteins inhibit the development of dimethylhydrazine-induced malignancy Papenburg R Bounous G Fleiszer D Gold P Tumour Biol 1990 11 129 136 2343238 Soybeans, soy foods, isoflavones and risk of colorectal cancer: a review of experimental and epidemiological data Toyomura K Kono S Asian Pac J Cancer Prev 2002 3 125 132 12718590 Soy consumption and colorectal cancer Spector D Anthony M Alexander D Arab L Nutr Cancer 2003 47 1 12 10.1207/s15327914nc4701_1 14769532 Microarray gene analysis of the liver in a rat model of chronic, voluntary alcohol intake Deaciuc IV Peng X D'Souza NB Shedlofsky SI Burikhanov R Voskresensky IV de Villiers WJ Alcohol 2004 32 113 127 10.1016/j.alcohol.2003.12.001 15163562 Gene expression profile in the livers of rats orally administered ethinylestradiol for 28 days using a microarray technique Kato N Shibutani M Takagi H Uneyama C Lee KY Takigami S Mashima K Hirose M Toxicology 2004 200 179 192 10.1016/j.tox.2004.03.008 15212814 Cancer Cell Cycles Charles JS Science 1996 274 1672 1677 10.1126/science.274.5293.1672 8939849 β-Catenin regulates expression of cyclin D1 in colon carcinoma cells Tetsu O McCormick F Nature 1999 398 422 426 10.1038/18884 10201372 Antisense to cyclin D1 inhibits the growth and tumorigenicity of human colon cancer cells Arber N Doki Y Han EK-H Sgambato A Zhou P Kim N-H Delohery T Klein MG Holt PR Weinstein IB Cancer Res 1997 57 1569 1574 9108461 Cyclin D1 is essential for neoplastic transformation induced by both E6/E7 and E6/E7/ErbB-2 cooperation in normal cells Al Moustafa AE Foulkes WD Wong A Jallal H Batist G Yu Q Herlyn M Sicinski P Alaoui-Jamali MA Oncogene 2004 23 5252 5256 10.1038/sj.onc.1207679 15229656 The neu oncogene: more than a prognostic indicator? De Potter CR Hum Pathol 1994 25 1264 1268 10.1016/0046-8177(94)90083-3 8001919 Expression pattern of the neu (NGL) gene-encoded growth factor receptor protein (p185neu) in normal and transformed epithelial tissues of the digestive tract Cohen JA Weiner DB More KF Kokai Y Williams WV Maguire HC Jr LiVolsi VA Greene MI Oncogene 1989 4 81 88 2563577 Intestinal tumor progression is associated with altered function of KLF5 Bateman NW Tan D Pestell PG Black JD Black AR J Biol Chem 2004 279 12093 12101 10.1074/jbc.M311532200 14726538 Involvement of aquaporins in colorectal carcinogenesis Moon C Soria JC Jang SJ Lee J Obaidul Hoque M Sibony M Trink B Chang YS Sidransky D Mao L Oncogene 2003 22 6699 6703 10.1038/sj.onc.1206762 14555983 Localization of mucin (MUC2 and MUC3) messenger RNA and peptide expression in human normal intestine and colon cancer Chang SK Dohrman AF Basbaum CB Ho SB Tsuda T Toribara NW Gum JR Kim YS Gastroenterology 1994 107 28 36 8020672 Role of gastrointestinal hormones in the proliferation of normal and neoplastic tissues Thomas RP Hellmich MR Townsend CM Jr Evers BM Endocr Rev 2003 24 571 599 10.1210/er.2002-0028 14570743 Antiproliferative effect of somatostatin and analogs Bousquet C Puente E Buscail L Vaysse N Susini C Chemotherapy 2001 47 Suppl 2 30 39 10.1159/000049159 11275700 The antitumor activity of the somatostatin structural derivative (TT-232) on different human tumor xenografts Tejeda M Gaal D Barna K Csuka O Keri G Anticancer Res 2003 23 4061 4066 14666719 Somatostatin inhibits tumor angiogenesis and growth via somatostatin receptor-3-mediated regulation of endothelial nitric oxide synthase and mitogen-activated protein kinase activities Florio T Morini M Villa V Arena S Corsaro A Thellung S Culler MD Pfeffer U Noonan DM Schettini G Albini A Endocrinology 2003 144 1574 1584 10.1210/en.2002-220949 12639942 Lack of peptide YY immunoreactivity in adenomatous colonic polyps: evidence in favor of an adenoma-carcinoma sequence Adrian TE Ballantyne GH Zucker KA Zdon MJ Tierney R Modlin IM J Surg Res 1988 44 561 565 10.1016/0022-4804(88)90162-X 3374119 Neuroendocrine cells within colorectal tumours induced by dimethylhydrazine. An immunocytochemical study Johnston CF O'Neill AB O'Hare MM Buchanan KD Cell Tissue Res 1986 246 205 210 10.1007/BF00219019 3779800 Colonic endocrine cells in rats with chemically induced colon carcinoma Sitohy B El-Salhy M Histol Histopathol 2001 16 833 838 11510974 Peptide YY as a growth factor for intestinal epithelium Mannon PJ Peptides 2002 23 383 388 10.1016/S0196-9781(01)00615-5 11825653 Glucagon enhances growth of cultured human colorectal cancer cells in vitro Moyer MP Aust JB Dixon PS Levine BA Sirinek KR Am J Surg 1985 150 676 679 10.1016/0002-9610(85)90406-4 4073359 Emergence of CD52-, glycosylphosphatidylinositol-anchor-deficient lymphocytes in rheumatoid arthritis patients following Campath-1H treatment Brett SJ Baxter G Cooper H Rowan W Regan T Tite J Rapson N Int Immunol 1996 8 325 334 8671618 The three Es of cancer immunoediting Dunn GP Old LJ Schreiber RD Annu Rev Immunol 2004 22 329 360 10.1146/annurev.immunol.22.012703.104803 15032581 T-cell receptor signal transmission: who gives an ITAM? Pitcher LA van Oers NS Trends Immunol 2003 24 554 560 10.1016/j.it.2003.08.003 14552840 Alterations in signal transduction molecules in T lymphocytes from tumor-bearing mice Mizoguchi H O'Shea JJ Longo DL Loeffler CM McVicar DW Ochoa AC Science 1992 258 1795 1798 1465616 Alterations of gene expression during colorectal carcinogenesis revealed by cDNA microarrays after laser-capture microdissection of tumor tissues and normal epithelia Kitahara O Furukawa Y Tanaka T Kihara C Ono K Yanagawa R Nita ME Takagi T Nakamura Y Tsunoda T Cancer Res 2001 61 3544 3549 11325815 Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by oligonucleotide arrays Notterman DA Alon U Sierk AJ Levine AJ Cancer Res 2001 61 3124 3130 11306497 Osteopontin identified as lead marker of colon cancer progression, using pooled sample expression profiling Agrawal D Chen T Irby R Quackenbush J Chambers AF Szabo M Cantor A Coppola D Yeatman TJ J Natl Cancer Inst 2002 94 513 521 11929952 Gene expression in colorectal cancer Birkenkamp-Demtroder K Christensen LL Olesen SH Frederiksen CM Laiho P Aaltonen LA Laurberg S Sorensen FB Hagemann R Orntoft TF Cancer Res 2002 62 4352 4363 12154040 Identification and validation of genes involved in the pathogenesis of colorectal cancer using cDNA microarrays and RNA interference Williams NS Gaynor RB Scoggin S Verma U Gokaslan T Simmang C Fleming J Tavana D Frenkel E Becerra C Clin Cancer Res 2003 9 931 946 12631590 AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition <it>ad hoc </it>writing committee on the reformulation of the AIN-76A rodent diet Reeves PG Nielsen FH Fahey GC Jr J Nutr 1993 123 1939 1951 8229312 Sources of variability and effect of experimental approach on expression profiling data interpretation Bakay M Chen YW Borup R Zhao P Nagaraju K Hoffman EP BMC Bioinformatics 2002 3 4 65691 11936955 10.1186/1471-2105-3-4 Significance analysis of microarrays applied to the ionizing radiation response Tusher VG Tibshirani R Chu G Proc Natl Acad Sci USA 2001 98 5116 5121 33173 11309499 10.1073/pnas.091062498 Expression profiling in the muscular dystrophies: identification of novel aspects of molecular pathophysiology Chen Y Zhao P Borup R Hoffman EP J Cell Biol 2000 151 1321 1336 10.1083/jcb.151.6.1321 11121445