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This is to certify that the
dissertation entitled

BLACK BEANS AND SOY FLOUR INHIBIT EXPERIMENTAL
COLON CARCINOGENESIS BY REDUCING INFLAMMATION AND
ENHANCING COLONOCYTE DIFFERENTIATION

presented by

ELIZABETH ANN RONDINI

has been accepted towards fulfilIment
of the requirements for the

Doctoral degree in Human Nutrition

 

 

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2/05 p:lClRC/DateDue.indd-p.1

BLACK BEANS AND SOY FLOUR INHIBIT EXPERIMENTAL COLON
CARCINOGENESIS BY REDUCING INFLAMMATION AND ENHANCING
COLONOCYTE DIFFERENTIATION
By

Elizabeth Ann Rondini

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Food Science and Human Nutrition

2006

ABSTRACT
BLACK BEANS AND SOY FLOUR INHIBIT EXPERIMENTAL COLON
CARCINOGENESIS BY REDUCING INFLAMMATION AND ENHANCING
COLONOCYTE DIFFERENTIATION
By
Elizabeth Ann Rondini

Colorectal cancer (CRC) is one of the most common neoplasms afflicting
Western societies (1). The geographical differences in CRC and studies in migrant
populations provide strong evidence that a majority of sporadic CRC cases are
attributable to environmental exposures, more specifically dietary habits (2-4). Legumes
(peas, beans, peanuts, lentils) are among dietary components frequently consumed by
populations who have a lower risk for cancers of the colon, breast, and prostate (7).
Experimental studies conducted in animals have also demonstrated that bean-based diets
inhibit the development of colonic neoplasms (14-18, 265). Because diet is a major
determinant of risk for CRC, this research was designed to achieve a more thorough
understanding of cellular and molecular events involved in chemoprevention by beans
using the azoxyrnethane (AOM)-induced rodent model of colon cancer.

The first experiment was conducted to assess biological processes involved in
cancer promotion. Microarrays were performed to profile genes differentially expressed
in colon tumors compared to non-neoplastic colonic mucosa in rats treated with the
carcinogen AOM. A majority of genes affected during cancer development were related
to immune responses and inflammation, extracellular matrix remodeling, and suggestive

of global disturbances in cellular metabolism and epithelial ion transport. Having

determined genetic alterations associated with cancer promotion, the second experiment

was designed to identify molecular targets permissive for tumorigenesis and those
associated with cancer inhibition by bean-feeding. Microarrays were performed on
mRNA isolated from normal-appearing colonic mucosa in rats treated with either AOM
or saline and fed a control (casein), black bean (BB), or soy-flour (SF) based diet for 30
weeks. Carcinogen (AOM) treatment was found to induce expression of genes involved
in inflammatory and immune responses, protein synthesis, and extracellular matrix
remodeling. BB- and SF-fed rats exhibited a higher expression of genes involved in
energy metabolism and a lower expression of inflammatory and cell cycle-associated

genes. Genes involved in extracellular matrix (collagen lotl, fibronectin 1) and in innate

immunity (NP defensin 3a, secretory phospholipase A2 (sPLA2)) were induced by AOM
in all diets, but less so in bean-fed animals. This gene profile suggests that bean diets
inhibit colon cancer by maintaining crypt cell homeostasis and reducing inflammation.
The third study was designed to address whether dietary differences in sPLA2
were associated with alterations in colonic epithelial proliferation and inflammation in
colon tissue using immunohistochemical techniques. Rats treated with AOM and fed a
control diet were found to have a higher zone of proliferative cells, a higher content of
sPLA2, and evidence of infiltrating macrophages in colonic tissue compared to rats fed
BB, SF, or control—fed animals not administered carcinogen. A positive and significant
correlation was found between sPLA2 immunoreactivity and the zone of proliferative
cells. These data, consistent with microarray profiles suggest that the ability of beans to
suppress colon cancer occurs early in the neoplastic process and is associated with

modulating inflammation and enhancing cellular differentiation.

 

 

 

DEDICATION

T o my family, especially my parents, for their love, encouragement, and support at all

times. Thank you for everything... you mean the world to me.

iv

 

ACKNOWLEDGEMENTS

I would like to thank my major advisor, Dr. Bennink, for his support and guidance
throughout my graduate studies. I would also like to extend my gratitude to Dr. Romsos
and my committee members, Dr. Bourquin, Dr. Claycombe, and Dr. Orth, for their useful
suggestions and review of my dissertation, and to Annette Thelen for technical assistance
with microarrays. I would also like to thank my parents for their support, both
emotionally and financially, which has made my education both possible and meaningful.
Lastly a “shout out” to my best fi'iends Joy Dubost and Mary Przepiora, sister Nicole
“Bangs”, and friends and colleagues Kathleen Barrett, Janette Harkins, Inese Cakstina,
Neil Birmingham, and Bing Wang for their assistance with my research, encouragement,

and for all the good times.

 

 

TABLE OF CONTENTS

LIST OF TABLES ............................................................................ viii
LIST OF FIGURES ........................................................................... x

I. INTRODUCTION ....................................................................... 1
11. REVIEW OF LITERATURE .......................................................... 4

A. INCIDENCE AND ETIOLOGY OF COLORECTAL CANCER. 5

B. PATHOGENESIS OF COLORECTAL CANCER 6

3.1 Normal histology ......................................................... 6

B2 Histopathology ........................................................... 6

B3 Molecular genetics ....................................................... 8

BA Inflammation and NSAIDs ............................................. 13

B.5 Carcinogen-induced model ofcolon cancer. . 15

C. DIETARY FACTORS AND COLORECTAL CANCER .................. 16

D. LEGUMES AND COLORECTAL CANCER ................................ 23

DJ Epidemiological studies ................................................. 25

D2 Experimental studies .................................................... 27

D3 Non-nutritive bioactive compounds ................................... 29

E. RATIONALE ...................................................................... 32

F. LITERATURE CITED .......................................................... 35
1H. PROFILE OF GLOBAL GENE PATTERNS ALTERED IN
AZOXYMETHANE (AOM)-INDUCED COLON TUMORS COMPARED TO

NORMAL-APPEARING MUCOSA ...................................................... 62

A. ABSTRACT ....................................................................... 63

B. INTRODUCTION ................................................................ 64

C. MATERIALS AND METHODS ............................................... 65

D. RESULTS .......................................................................... 70

E. DISCUSSION ..................................................................... 81

F. LITERATURE CITED .......................................................... 85

IV. MICROARRAY ANALYSES OF GENES DIFFERENTIALLY
EDRESSED BY DIET (BLACK BEANS AND SOY FLOUR) DURING 9O
AOM-INDUCED COLON CARCINOGENESIS IN RATS ...................
A. ABSTRACT ....................................................................... 91

vi

 

 

B. INTRODUCTION ................................................................ 92
C. MATERIALS AND METHODS ............................................... 93
D. RESULTS ........................................................................... 100
E. DISCUSSION ..................................................................... l 16
F. LITERATURE CITED .......................................................... 123

V. BLACK BEANS AND SOY FLOUR REDUCE INFLAMMATORY
BIOMARKERS AND RESTORE COLONOCYTE DIFFERENTIATION

 

DURING AOM-INDUCED COLON CARCINOGENESIS IN RATS... . . . . 132 r‘
A. ABSTRACT ........................................................................ 133
B. INTRODUCTION ................................................................ 134
C. MATERIALS AND METHODS ............................................... 135
D. RESULTS .......................................................................... 1 39
E. DISCUSSION ..................................................................... 146
F. LITERATURE CITED .......................................................... 149
VI. SUMMARY AND CONCLUSIONS .................................................. 152
VII. FUTURE DIRECTIONS ................................................................ 155
VIII. APPENDICES ......................................................................... 157

vii

 

LIST OF TABLES

CHAPTER II.
Potential mechanisms relating diet and nutrition-related factors

TABLE 1. to CRC risk .............................................................. 18
Nutrient composition of common beans and soy products per

TABLE 2. 100 g ....................................................................... 24

CHAPTER III.

TABLE 1. Primer pairs used for RT-PCR ......................................... 69
Genes significantly altered in AOM-induced colon cancers

TABLE 2. relative to normal colonic mucosa .................................... 73

CHAPTER IV.

TABLE 1. Nutrient composition of experimental diets ........................ 95

TABLE 2. Primer pairs used for RT-PCR ...................................... 98
Select genes differentially expressed by carcinogen (AOM)

TABLE 3. treatment in the distal colonic epithelium of male F344 rats... 104

Genes similarly affected by bean-feeding (BB and SF) in the
TABLE 4. distal colonic epithelium of male F344 rats. ..................... 107

Genes significantly affected by carcinogen (AOM) injection
and dietary treatment in the distal colonic epithelium of male
TABLE 5. F344 rats ............................................................... 113

CHAPTER V.

Main effects of treatment on PCNA proliferation indices and
sPLA2 intensity in the distal colonic epithelium of male F44
TABLE 1. rats ..................................................................... 141

viii

 

APPENDICES.

SUPPLEMENTARY

TABLE

SUPPLEMENTARY
TABLE

SUPPLEMENTARY
TABLE

Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal

colonic mucosa ............................................ 158
Supplemental list of genes differentially affected by
AOM treatment in distal colonic epithelium of male

F344 rats .................................................. 170
Supplemental list of genes differentially expressed

by dietary treatment in distal colonic epithelium of

male F 344 rats ............................................ 177

ix

LIST OF FIGURES

CHAPER II.

FIGURE 1. Genetic alterations in colon cancer .......................................... 9

CHAPTER III.

Biological classification of genes altered in AOM-induced colon

 

FIGURE 1. cancers ....................................................................... 72
RT-PCR analysis of genes differentially expressed in AOM-
FIGURE 2. induced colon cancers ...................................................... 80
CHAPTER IV.

Biological classification of known transcripts significantly altered
in the distal colonic epithelium of male F344 rats as affected by
FIGURE 1. AOM-injections and dietary (AIN, BB, SF) treatment (P < 0.05)... 102

RT-PCR analysis of cell cycle-associated genes differentially

FIGURE 2. affected by dietary treatment ................................................ 114
Relative fold-changes in sPLA2 mRNA abundance detected with
FIGURE 3. RT-PCR ........................................................................ 1 15
CHAPTER V.
FIGURE 1. Immunoreactivity for sPLA2 in colonic mucosa ........................ 142

Correlation between PCNA proliferation zone (highest PCNA”
FIGURE 2. cell/crypt height) and sPLA2 immunoreactivity in colonic crypts. . .. 143

Percent area of distal colonic lamina propria covered by CD68
FIGURE 3. macrophages. ............................................................... 144

FIGURE 4. Immunoreactivity for CD68+ macrophages in colonic mucosa... 145

 

 

CHAPTER I.

INTRODUCTION

 

Colorectal cancer (CRC) is the one of the most common neoplasms afflicting
industrialized societies (1). In 2002, there were 528,978 deaths due to colorectal cancer
worldwide, with 59,345 cases in the United States alone (1). Both genetic and
environmental exposures have been implicated in the etiology of CRC, and up to 75% of
cases may be preventable by adequate diets and regular exercise (2-4). Generally,
consumption of diets low in red meat and alcohol and high in vegetables and cereal
grains is associated with a decreased risk of developing CRC (2-4). Results from several
epidemiological studies also indicate that populations consuming higher intakes of
legumes (peas, beans, lentils, peanuts) have a lower occurrence of (5-6) and mortality
from CRC (7). Legumes are naturally high in dietary fiber, micronutrients, and several
bioactive compounds (te. phytoestrogens, protease inhibitors, saponins, phytic acid) that
may contribute to anti-cancer properties (270-276, 280-281). However the mechanisms
underlying reduced susceptibility to colon cancer development have not been fully
elucidated.

The development of colon cancer from normal epithelium is a genetically driven
process (8-11). Several genes altered during the adenoma-carcinoma sequence in humans
have been identified (8-12), and have led to a more thorough understanding of molecular
events important in the pathogenesis of CRC. With the advent of technology available to
explore multiple genes simultaneously, it is now possible to determine how certain foods
and/or food components influence colon carcinogenesis at a cellular level.

Several animal models have been developed to examine various aspects of colon
cancer under controlled conditions (13). Among these, the azoxymethane (AOM)-

induced rodent model is one of the most widely used models for examining dietary

 

 

influences on colon cancer development. In this model, feeding either dry beans (14-15)
or soy flour (16-18) inhibits the development of carcinogen-induced colon tumors.
Therefore, the purpose of the current research was to identify early cellular events
associated with colon cancer inhibition by beans in vivo using oligonucleotide
microarrays. Histochemical procedures were then performed to assess diet-related

differences in colonic epithelial kinetics and inflammation.

CHAPTER II.

LITERATURE REVIEW

A. INCIDENCE AND ETIOLOGY 0F COLORECTAL CANCER.

Colorectal cancer (CRC) is one of the most common types of neoplasms, with an
estimated total 1,023,152 cases diagnosed worldwide in 2002 (l). The highest rates
occur in more developed regions such as North America, Western Europe, and Australia
contributing to 65% of the total global incidence of CRC (1,2). In the United States,
CRC remains the fourth most common type of cancer and, after lung cancer, is the
second leading cause of all cancer deaths (19). Incidence rates are slightly higher in men
than women, with a predominance of cancers arising in the distal colon (30%) and rectum
(29%) and the remainder (39%) in the proximal colon (20).

Both genetic and environmental factors have been implicated in the etiology of
CRC. The inherited syndromes familial adenomatous polyposis (FAP) and heredity non-
polyposis colorectal cancer (HNPCC) account for approximately 5-10% of cases and up
to 25% may arise from genetic susceptibility factors that have not yet been fully
elucidated (21). The remainder of cases (65-70%) arise sporadically, with over 90% of
new CRC cases diagnosed in individuals greater than 50 years of age (11, 19, 21). The
geographical differences in CRC and studies in migrant populations provide strong
evidence that a majority of sporadic cancers are attributable to environmental exposures
(2). More specifically, dietary and lifestyle habits are thought to contribute to most of the
worldwide variation in the incidence of CRC (2). Generally, populations at increased
risk for the disease consume higher intakes of red and processed meat and alcohol and
lower intakes of cereals, dietary fiber, and vegetables (2-4). Other factors related to an
increased risk include obesity (4, 22-25), insulin resistance (25-28), and ulcerative colitis

(29) whereas physical activity (4, 30), nonsteroidal anti-inflammatory drugs (NSAIDs)

(4, 31), and, in women, hormone replacement therapy are inversely associated with CRC

(4, 32).

B. PATHOGENESIS OF COLORECTAL CANCER.
B_.l Normal Histology.

The human colon is approximately 150 cm (5 ft.) in length and is functionally
important for water and electrolyte absorption and evacuation of stool (33-34). The
surface of the colon is composed of a single layer of epithelial cells, termed the mucosa,
that extend downward into the lamina propria forming crypts (35-36). The epithelial
lining is maintained by a population of multipotent stem cells anatomically located in the
base of the crypts (35-36). Cell division is normally confined to lower 1/3 of the crypts
and as cells migrate towards the lumen, they terminally differentiate into absorptive,
goblet, or enteroendocrine cell types (35-36). Once cells reach the crypt apex, they
undergo apoptosis and are shed into the lumen. In humans, the entire surface of the colon

is replaced every 3-6 days (35).

_I_3._2_ Histopathology.

The development of colorectal cancer from normal appearing epithelium is
associated with alterations in cell proliferation, differentiation, apoptosis, and cellular
senescence. These changes are genetically driven and for a majority of CRC (>90%)
manifest through distinct histological steps (37-39). An early event in the process of
colon carcinogenesis involves an expansion of the proliferative zone towards the lumen
(37-38). This hyperproliferative change precedes tumor development and has been

identified throughout the colon of humans predisposed to colon cancer and in rodents

 

 

administered carcinogen (37, 40). Eventually, either through gene mutation or deletion,
there is a focal defect resulting in an abnormal accumulation of proliferative cells (polyp)
within a crypt. Two types of polyps can be distinguished histologically. Hyperplastic
(nondysplastic) polyps are more common, containing cells with normal morphology
aligned in a single layer along the basement membrane, and generally not considered to
have malignant potential (35). Comparably, adenomatous (dysplastic) polyps contain
epithelial cells with abnormal morphology, arranged in several layers along the basement
membrane, and are more likely to progress to cancer (35). As adenomatous polyps grow
in size the epithelial lining invaginates and expands in various directions, forming first a
tubular than villus structure (11, 35, 38). Transition to malignancy is accompanied by a
progressive increase in dysplasia followed by the potential of cells to invade the
basement membrane and metastasize to distant organs (11, 35, 38).

Although polyps are the earliest macroscopic lesions detectable, aberrant crypt
foci (ACF) have been proposed to precede adenoma development. ACF are microscopic
lesions usually composed of crypts that are larger in size, have altered lumenal openings,
and exhibit thickened epithelia (41-43). ACF are found in colons of rodents injected with
carcinogen and in humans at increased risk of colorectal cancer. Although ACF contain
similar genetic alterations to those in human cancers, a large portion of ACF are not
dysplastic (42) and, in animals, ACF do not consistently correlate with adenocarcinoma
development (43-44). Therefore the proposed role of ACF as precursors to adenomas

remains elusive.

13;; Molecular genetics.

Carcinogenesis is considered to be a multi-step process, with each step
representing mutations, deletions, and/or epigenetic alterations in genes normally
regulating cell growth and behavior (1 1). Two classes of genes are commonly affected in
neoplasia. Tumor suppressor genes normally restrain cell growth and inactivation of both
alleles results in a “loss of function” phenotype. Conversely, proto-oncogenes promote
normal cell growth and mutations cause constitutive activation (45). Each mutation
confers the cell with a selective growth advantage accompanied by a progressive change
from normal to malignant behavior (1 l, 46). For each tissue, the mutations and order that
they occur comprise the genetic pathway (11).

More than 10 years ago, Fearon and Vogelstein proposed the genetic model for
colorectal cancer development based on mutational analyses of tumors at various stages
of the adenoma-carcinoma sequence (8-10). According to this model, colorectal cancer
arises from the cumulative loss of functional tumor suppressor genes and dominant
activation of proto-oncogenes, with the former predominating (9-10). Somatic mutations
in at least 4-5 genes are necessary for malignant transformation, whereas fewer are
associated with benign tumor growth. Further, genetic alterations occur in a preferred
sequence, with only a subset of genes capable of initiating the tumorigenic process (8-
10). The most common mutational events include loss of function of the adenomatous
polyposis coli (APC) gene, p53 gene, one or more genes on chromosome 18q, and

constitutive activation of k-ras (FIGURE 1).

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APC, located on chromosome 5q21 in humans, encodes a protein with tumor
suppressive properties (10). Inactivation of APC, either through mutation or allelic loss
is permissive for adenoma formation and has been detected in 60-80% of sporadic colon
tumors (47-48). Analysis of the APC gene from sporadic and inherited forms of colon
cancer indicate that mutations occur most frequently in codons 1286-1513, named the
mutation cluster region (MCR) (48-50). More than two-thirds of mutations result in
frameshift, and one-third involve nonsense point mutations, both resulting in premature
truncation of the polypeptide (49). Functional regions downstream of the MCR include
those involved in down regulation of B-catenin and binding sites for interaction with
microtubules, E81, and DLG, suggesting these regions may all be involved in the tumor
suppressive properties of APC (49, 50).

The involvement of APC in controlling intracellular levels of B-catenin has been
one of the most widely researched areas in colon tumorigenesis. B-catenin associates
with cadherins in the plasma membrane and also exists in a monomeric (free) form in the
cytoplasm, which, upon stimulation, functions in a signal transduction pathway (51, 52).
In the absence of Wnt signaling, the cytosolic level of B-catenin is tightly controlled (45).
Glycogen synthase kinase (GSK) 30, a serine/threonine kinase, phosphorylates both [3-
catenin and APC (45, 49, 53) and phosphorylation results in an APC/B-catenin/GSK-
3B/axin complex that mediates degradation of B-catenin by the ubiquitin-proteosome
pathway (53, 54). Initiation of the Wnt/wingless signal transduction pathway, which

inhibits GSK 3B, or mutations in the B-catenin binding site on APC, both result in

stabilization of cytosolic B-catenin, enabling it translocate into the nucleus, where it

10

 

 

interacts with a family of high mobility group-box (HMG) containing transcription
factors (TCF-l, TCF-2, TCF-4, LEF-l) and initiates transcription of genes (45, 51, 52,
55-56). A few of the target genes have been found to be those involved in the cell cycle
and tumor progression, including c-Myc (57), cyclin D1 (58), c-Jun (59), survim'n (60),
matrilysin (61), fibronectin (62) and PPAR5 (63).

Thus, the regulation of cytoplasmic levels of B-catenin is likely an important
function of APC. This is further corroborated by the finding that gain-of-function
mutations in the NH2 regulatory domain of B-catenin occurs in 48% of human colorectal
tumors lacking APC mutations (64). However, important tumor suppressive properties of
APC are also likely mediated through interactions with other cellular proteins. Some of
these events involve apoptosis, cytoskeletal reorganization, chromosomal stability, and

cell migration and mobility (reviewed in 50).

221-2.- 1.91%

The k—ras gene, located on chromosome 12p encodes a 21-kDa, guaninine
nucleotide binding protein with intrinsic GTPase activity (35). The ras proteins (k-ras,
N-ras, and H-ras) are involved in transducing mitogenic signals from receptor tyrosine
kinases in response to growth factors, cytokines, and hormones (35, 65). Binding of
GTP activates ras whereas GTP hydrolysis, facilitated through GTPase activating
proteins (GAPS), leads to the inactive, GDP-bound form (65). Ras oncogenes are made
constitutively active through point mutations occurring at amino acids 12, 13, or 61,

rendering them insensitive to GAPS (35, 65). Mutations in k-ras generally occur later in

11

adenoma growth and have been detected in 50% of colorectal carcinomas and in

adenomas larger than 1 cm in size (35).

g3; QCC, QPC4ISmad4z, and JV18-1(Smad22.

One copy of chromosome 18q is lost in 47% of advanced adenomas and 73% of
adenocarcinomas, but the target gene(s) has not been conclusively determined (11, 35).
One candidate is deleted in colon cancer (DCC), which encodes a large transmembrane
protein that contains immunoglobulin-like domains and shares homology with neural
adhesion molecules (11, 35). The protein is expressed in a variety of tissues at points of
cell-cell contact, suggesting a role in differentiation (66). Two other candidate genes
include deleted in pancreatic carcinoma (DPC4, or Smad4) and JV18-1 (also called
SmadZ) (35). Both of these proteins function downstream in the transforming grth

factor (TGF)-0 signaling pathway (11, 35). TGFB exerts an inhibitory effect on cell

growth in normal colorectal epithelium (67).

343:! L53.-

Mutational inactivation of the p53 gene occurs late in tumor progression, most
likely in the adenoma-carcinoma transition (9-10, 35). p53 normally functions to mediate
anti-proliferative processes and/or apoptosis in response to cellular stresses (reviewed in
68, 69). Genomic damage, hypoxia, and aberrant oncogene expression stabilize p53,
allowing transactivation of genes such as p21 WAF/C’P (69-72) and Growth Arrest and

DNA-Damage Induced (GADD) 45a (69-70, 73). These proteins block progression

through the cell cycle and allow repair of DNA damage by inhibiting the activity of

12

cyclin dependent kinases (68, 69). p53 also mediates apoptosis in response to cellular
damage through transcription-dependent and independent mechanisms, but the signaling

pathways involved have not been completely elucidated (68, 69).

B.3.5 Epigenetic events.

In addition to the model proposed by Fearon and Vogelstein, aberrant methylation
patterns are frequently observed in colorectal cancers. Methylation of 5’cytosine residues
in gene-specific promoter regions is a naturally occurring modification of DNA that is
heritable (74). Hypermethylation of certain promoter regions leads to transcriptional
silencing whereas hypomethylation can lead to transcriptional activation. In both benign
and malignant colon tumors global hypomethylation of DNA is frequently observed (75).
In addition, promoter-specific hypermethylation in genes including the tumor suppressors
APC, p16, and p14”F and those involved in mismatch repair have been reported (74).
These changes would likely contribute to cancer by affecting cell cycle regulation and
capability of DNA repair, the latter leading to a “mutator” phenotype observed in a subset

of tumors.

BA, Inflammation/NSAIDS.

Although specific genetic mutations are required for initiation and propagation of
tumorigenesis, modifier genes and/or the tumor microenvironment can also substantially
contribute to colon cancer development. Inflammation has long been recognized to be
involved in the etiology of CRC. Patients with a history of recurrent ulcerative colitis are

at an increased risk of developing the disease (29). Additionally, Waddell et a1. (76)

13

recognized over 25 years ago that administering the NSAID sulindac to patients with the
inherited condition familial adenomatous polyposis (FAP) caused a significant regression
of rectal polyps. Since then, several studies in humans (reviewed in 77) and animals (7 8-
84) have established that NSAIDs inhibit colorectal tumorigenesis. In a review of
published studies, the authors estimated a 40-50% reduction in sporadic colon adenomas,
cancer, and mortality for regular users of NSAIDs (77).

The anti-neoplastic properties of NSAIDs have not been fully established. They
bind to and inhibit the activity of cyclooxygenase (COX) enzymes, which catalyze the
conversion of arachidonic acid to prostaglandins (PG) and thromboxane (TX) (85). The
gastrointestinal tract contains two isoforrns of COX. COX-l is constitutively expressed
and important for normal physiological processes such as maintenance of the
gastrointestinal mucosa whereas COX-2 is normally expressed at very low levels but can
be induced by pro-inflammatory cytokines, grth factors, lipopolysaccharides (LPS),
and mitogens (85).

Treatment of cancer cells with NSAIDS increases apoptosis, induces cell-cycle
arrest, and inhibits angiogenesis, presumably through down-regulating the synthesis of
prostaglandins (85-89). Because increased levels of COX-2 have been detected in up to

APC mice (84), and in

90% of human colon cancer tissues (85), in adenomas from Min
colon tumors from rodents induced with carcinogen (90), most of the anti-neoplastic
effects of NSAIDS are thought to be mediated by this isoforrn. Boolbol et al (84)
reported elevated COX-2 and PGE2 in Min”C mouse intestines compared wild-type

littennates. Treatment of mice with sulindac inhibited tumor formation, reduced both

COX-2 protein and PGE2 levels, and restored enterocyte apoptosis. More direct evidence

14

 

 

for involvement of COX—2 in tumorigenesis was demonstrated by Oshima et al (91) who
reported targeted disruption of COX-2 in ApcA716 knockout mice reduced polyp
formation by ~86% and also reduced polyp size (91). However, NSAIDs that are not
selective for COX-2 such as aspirin and sulindac sulphone are also as effective in
inducing apoptosis and suppressing tumorigenesis in viva, suggesting that some of the
anti-neoplastic effects of NSAIDs may be mediated through PG-independent pathways

(92-94).

L5; Carcinogen-induced model of colon cancer.

Although CRC was one of the first cancers to be characterized in terms of a
genetic model, examining the etiology of carcinogenesis in humans is hampered due to
the lack of specific prognostic biomarkers, heterogeneity among population groups, and
the latency of time it takes for colorectal tumors to manifest. The use of animal models
has proven a useful tool for studying various aspects of human disease under controlled
laboratory conditions. Druckery and colleagues were the first to demonstrate that
administration of the synthetic compound 1,2-dimethylhydrazine (DMH) or one of its
metabolites azoxymethane (AOM) specificity and reliably induced colon tumors in
rodent species (95-97). Following administration, DMH and AOM are metabolized in
the liver to methyldiazonium (MDM) (97). MDM is an active methylating agent that
forms a number of DNA adducts in vivo, with 06-methyldeoxyguanosine (06-mdg) being
one of the major mutagenic DNA lesions produced (98). Prior to replication, this lesion
can be repaired in a “suicide” fashion by Oé-alkylguanine-DNA alkytransferases (06-

atase) or removed by targeted apoptosis (98). The inability to remove 06-mdg by either

15

of these processes can cause G:C to A:T transitions during DNA replication, a common
genetic alteration reported in some human tumors (99).

The carcinogen-induced rodent model is one of the most widely used models for
studying colon carcinogenesis. Tumors develop almost exclusively in the colon,
primarily in the distal region, similar to the distribution observed in humans from high-
risk areas. Although APC mutations are rare in carcinogen-induced rodent colon tumors,
mutations in the GSK-3B phosphorylation consensus sites on B-catenin have been
reported (100). These sites are important for down-regulation of B-catenin by
ubiquitination and likely affect the stability of the protein. Takahashi et al. (100)
reported that in normal colon epithelium, B-catenin was primarily localized to the plasma
membrane, whereas a majority of dysplastic ACF, adenomas, and adenomacarcinomas
exhibited homogenous cytoplasmic and scattered nuclear staining. k-ras mutations on
codon 12 and 13 in DMH-induced colon adenocarcinomas have also been reported to
occur in a similar frequency to that observed in humans (101-102). In addition, local
inflammation, promoted in part through up-regulation of COX-2 and inducible nitric
oxide synthase (iNOS), is another common characteristic involved in the pathogenesis of

colon cancer from both species (103-104).

C. DIETARY FACTORS AND COLORECTAL CANCER.

Dietary habits are strongly linked to the etiology of CRC (2—4). Numerous
epidemiological studies have been conducted in attempts to identify specific dietary
components associated with higher risk of developing CRC or contributing to CRC
mortality (reviewed in 2-4, 105). Generally, data are most consistent for a lower

occurrence of CRC in individuals consuming higher intakes of vegetables, particularly

l6

raw, green, and cruciferous vegetables, and dietary fiber. Dietary components associated
with an increased risk include red meat and alcohol, and possibly dietary fat, processed
and heavily cooked meats, and sucrose (2, 4, 105). Metabolic consequences related to
over-nutrition, including obesity and insulin resistance have also been implicated in the
etiology of CRC (22-23, 106-112). Several hypotheses have been generated to explain
the association of diet and lifestyle habits with CRC and further tested in animal models.
There is currently no consensus as to how diet consistently modulates risk, which is
undoubtedly multi-factorial and likely depends on the ratio of both promoting and anti-
promoting factors. Some of the more established mechanisms are presented in TABLE 1
and are discussed below.

Red and processed meats may increase CRC by increasing lumenal concentrations
of potentially carcinogenic compounds (113-114). Heterocyclic amines (HCA),
produced by cooking meat at high temperatures are mutagenic in bacterial assays (115)
and dose-dependently induce large intestinal tumors when administered to rodents (116-
117). Meat consumption has also been associated with higher intakes of dietary fat.
Dietary fat and cholesterol stimulate bile acid secretion and populations at increased risk
for colon cancer excrete higher levels of bile acids in the stool (118). Approximately 2-
5% of bile acids escape reabsorption in the small intestine and are metabolized by colonic
microflora producing the secondary bile acids deoxycholate and litholicholic acid (119).
Secondary bile acids are irritating to the epithelium (120), induce cell proliferation (120),
and have been demonstrated to promote experimental colon cancer when administered to
animals (121-123). Adequate intakes of dietary calcium can form insoluble salts with

lumenal bile and fatty acids and calcium supplementation reduces hyperproliferation in

17

 

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18

the colorectal mucosa of rodents (124-125) and in humans at increased risk for CRC
(126-129).

Moderate alcohol consumption potentially increases CRC risk by influencing
mucosal cell proliferation (130-131), inhibiting DNA repair (132), and/or through
generation of DNA-acetylaldehyde adducts (132). The effect of alcohol on CRC risk
appears to be potentiated by insufficient dietary intake of folate or methionine (133-134).
Folate is required for normal functioning of the methionine cycle. Tetrahydrofolate
(THF) is the reduced form of folic acid capable of accepting one-carbon groups from
metabolites generated in amino acid metabolism and serves as a precursor for 5
coenzyme forms (135). These THF derivatives participate in distinct one-carbon
metabolism pathways including amino acid metabolism and purine and pyrimidine
synthesis (135-137). Ns-methyltetrahydrofolate (NS-methyl THF) regenerates methionine
from homocysteine, which can be converted to S-adenosyl-methionine (SAM). SAM is
required for biological methylation reactions, and may be reduced when diets are
deficient in labile methyl groups (137). This can result in DNA hypomethylation, which
may enhance oncogene expression (138). Aberrations in 5,10 methylene tetrahydrofolate
(a metabolite of folate) may also result in misincorporation of dUMP for dTMP during
DNA replication, thereby enhancing point mutations (136-137). In rapidly dividing cells
such as the intestinal epithelium, enzymatic mechanisms may not sufficiently repair DNA
damage prior to proliferation, causing heritable changes to be “fixed”. In support of this,
diets deficient in folate have been demonstrated to produce DNA strand breaks in the p53
gene in rat colon mucosa (139) and were associated with higher prevalence of k-ras

mutations in human colon adenomas (140-141). However, these diets contained sub-

19

optimal levels of dietary folate and it is not likely that intakes exceeding current
recommendations would provide any additional benefit to CRC.

Vegetables contain several antioxidant vitamins and minerals as well as bioactive
compounds that may contribute to reduced CRC risk (2, 142-151). Antioxidants may
inhibit carcinogenesis through scavenging reactive oxygen species (ROS) and preventing
ROS-induced DNA damage (152-156). Epidemiological and experimental data however
do not indicate that supplementation of antioxidant compounds beyond recommendations
is associated with reduced occurrence of CRC or colorectal polyps (157-163). Several
bioactive compounds present in vegetables have been suggested to exert anti-cancer
properties through a variety of mechanisms including induction of hepatic and intestinal
phase II detoxification enzymes (164-168), modulating oncogene activation (169-171),
and anti-inflammatory properties (172-176), among others (177-178). Vegetables and
cereal grains are also rich sources of dietary fiber, which is related to lower colon cancer
rates in some epidemiological studies (2, 179). However, dietary fibers differ in
physiochemical characteristics and identifying mechanisms by which fiber consistently
modulates colon carcinogenesis is difficult. Less fermentable fiber constituents and
sources including cellulose, lignins, and wheat bran may affect colon cancer by speeding
intestinal transit and diluting and adsorbing lumenal carcinogens or irritants. Bacterial
fermentation of more soluble fibers and non-starch polysaccharides lowers lumenal pH,
which can affect microbial metabolism and generation of secondary bile acids (180).
Fermentation also produces the short-chain fatty acids (SCFA) acetate, propionate, and
butyrate (181-182). Butyrate has been demonstrated to inhibit colon cancer cell growth

and induce apoptosis in vitro (183-185). Numerous animal studies have been conducted

20

to examine the influence of purified fiber sources on experimental colon carcinogenesis
and inconsistent results have been reported (186-198). Generally, wheat bran protects
against colon cancer more consistently than cellulose or more fermentable fiber sources
(187, 190-191, 194, 198). These results suggest that purified fibers alone do not appear
to inhibit tumorigenesis, whereas it is likely that some other factors present in vegetables
and grains and/or synergistic effects of fiber with other dietary components may be
responsible for anti-cancer properties.

Limited data are available for n-3 fatty acid consumption and CRC risk, although
animal studies have consistently shown a protective effect of fish oil on experimental
colon carcinogenesis (199-204). The inhibitory effects of fish oil on colon cancer have
been associated with enhancing cellular differentiation and apoptosis (200, 202),
suppressing the expression and/or activation of p21ras and COX-2 (205-207), reducing
protein kinase C (PKC) BII activity (208), and altering synthesis of prostaglandins (209-
210) and TXBz (210) in non-neoplastic mucosa.

Aside from specific dietary components, metabolic disorders related to over-
nutrition have been proposed to contribute to higher incidence of CRC. McKeown-
Eyssen (106) and Giovannucci (107) were the first to recognize that several risk factors
associated with CRC, such as sedentary lifestyles, obesity, and high fat diets were similar
to those for insulin resistance and diabetes. This led to the proposal that metabolic
consequences associated with insulin resistance (hyperinsulinemia, hyperglycemia,
hyper-triglyceridemia, increased non-esterified fatty acids (NEFAs)) promotes colon
carcinogenesis (106-107). Several possibilities for this association have been suggested.

First, high circulating levels of insulin may directly promote colonic tumorigenesis

21

directly through receptor binding and mitogenesis. Second, insulin resistance may
indirectly affect colon epithelial proliferation through increased intravascular energy
provision. A third model suggests that hyperinsulinemia may lead to increased insulin-
like growth factor-1 (IGF-l) bioavailability, a mediator of cell survival and growth, by
reducing hepatic IGF binding protein 1 (IGFBPI) production (211).

The epidemiology studies relating insulin resistance to colon cancer have been
recently reviewed (109). Most case-control and cross-sectional studies Show a slightly
elevated risk of CRC in diabetics, with odds ratios (OR) ranging from 1.0—2.9 (109).
Similar results have been reported using cohorts of patients with diabetes. However,
some of these studies are limited in interpretation because most relied on self-reporting of
diabetes and were not designed to address the extent of glycemic control. Prospective
studies evaluating biomarkers of insulin-resistance (i.e. C-peptide, HgBlc) rather than
history of diabetes generally show stronger positive associations, particularly for
advanced CRC and CRC mortality (109, 111-112). Increased risk for CRC has also been
reported in individuals with elevated plasma IGF-1 (112, 212) and in individuals with
acromegly, who have pathologic elevations of serum growth hormone and IGF—1 (pooled
OR=2.04) (213).

Colon cancer cells express both insulin (214) and IGF-1 receptors (IGF-1R) (215-
216), and treatment of cells with either insulin or IGF-l stimulates mitogenesis (217-
218). Additionally, IGF-1 up-regulates the expression of vascular endothelial growth
factor (VEGF) (219) and cell lines over-expressing IGF-1R are resistant to apoptosis and
are more metastatic than non-transfected cells (220). In animal studies, repeated

administration of insulin significantly increased the multiplicity of ACF (221) and the

22

number of colon tumors (222) in rats chemically induced with AOM, suggesting a direct
effect of insulin on tumor promotion. Koohestani et al. (223) conducted a study to
compare the effect of diet on insulin resistance and colon carcinogenesis and found that
high energy, high fat diets led to impaired glucose tolerance, which was positively

correlated with the size of ACF (r = 0.67, p<0.001).

D. LEGUMES AND COLORECTAL CANCER.

Legumes (peas, beans, lentils, peanuts) are one dietary component traditionally
consumed in populations where cancers of the colon, breast, and prostate are low (7), and
several of the mechanisms proposed to reduce CRC risk in humans and animals can be
achieved through increased consumption of legumes (TABLE 1). Legumes are unique
plant foods, providing a concentrated source of vegetable protein and a variety of both
essential and non-essential nutrients (224-226) (TABLE 2). Oilseeds, including
soybeans, and peanuts are higher in lipid content (24%) than dry beans (1-2%), but both,
are a significant source of complex carbohydrates and both soluble and insoluble dietary
fiber. Legumes are a good source of the minerals iron, calcium, copper, zinc, potassium,
and magnesium and the water-soluble vitamins thiamine, niacin, riboflavin and folate
(224-226). Several bioactive compounds including saponins, phytic acid, phenolics,
tannins, phytosterols, lectins and protease inhibitors are also present in legumes and some
have been shown to exert a number of health benefits (224-227).

In the US, soybeans are typically processed prior to consumption to generate
products with a higher protein content and to extract oil for commercial use (228).

Typically, soy products are grouped into three categories based on their protein content:

23

 

 

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24

soy flour, soy concentrates, and soy isolates. All three products are derived from defatted
soy flakes and range in crude protein content fi'om 50% (dry wt) in defatted soy flour to
>90% protein in soy isolates (TABLE 2). Soy flours and soy grits are the least processed
of the three and are made by grinding and screening soybean flakes (229). Soy
concentrates are prepared through aqueous or aqueous-ethanol extraction of defatted soy
flakes to remove soluble carbohydrate fractions. Isolates are the most refined soy protein
source through which most of the total carbohydrates from soy flour are removed. Soy
protein products are used in a variety of second-generation soy foods, bakery products,
breakfast cereals, and infant formulas, accounting for more than 90% of soybeans
consumed in the United States (228).

There has been renewed interest in studying legLunes with respect to chronic
disease prevention (225-227, 231-232). Several epidemiological and experimental
studies have implicated that incorporating more legumes into the diet has the potential to
aid in the prevention and/or management of several chronic diseases including diabetes
(225, 231-236), cardiovascular disease (6, 225-227, 231-232, 237-241), obesity (231-232,
242-244), and cancer (225, 227, 5-7). These diseases are more common in developed
countries, such as the United States, where consumption of beans and other plant

products remains below recommendations (225).

L1 Epidemiological Studies.

Most epidemiological studies examining relationships between diet and cancer
put little emphasis on legume/pulse consumption. In studies where legume intake is
assessed, distinctions are usually not made between the various legumes, making direct

correlations with colon cancer difficult. In two prospective studies examining dietary

25

patterns and disease risk as part of the Adventist Health Study in the US, significant
inverse associations between legume consumption and colon cancer were reported (5-6).
In the study by Singh and Fraser (5), individuals consuming legumes > 2 times/week
were 47% less likely to develop colon cancer when compared to individuals consuming
legumes never to < 1 time/week. Two case-control studies, one conducted in Australia
and the other in the Spanish island of Majorca, also reported a protective effect of legume
consumption on colon cancer risk. In the study by Steinmetz and Potter (245), the OR
was 0.4 and 0.7 for women and men with the highest legume (beans, split peas, lentils,
soybeans, chickpeas) intakes, respectively. Another case-control study in Majorca
reported on fiber from pulses, rather than as a group. Similar to the previous studies, the
authors reported a significant protective effect (P < 0.01) for individuals in the highest
quartile of legume fiber intake, with an OR of 0.4 (246). Correa (7) was the only study to
specifically examine bean consumption. Per capita data compiled from 41 countries,
revealed that countries with the greatest consumption of beans had the lowest mortality
rates due to breast (r = -0.70), prostate (r = -0.66), and colon (r = -0.68) cancer (7).

The epidemiological studies addressing soy intake and colorectal cancer risk have
been recently reviewed (247-248). A total of 13 studies (3 ecological, 1 cohort, and 9
case-control) have been conducted. In the three ecological studies examining soy intake
and mortality from colon or rectal cancer, 1 found a significant inverse association (249),
one found a positive association (250), and the third reported no significant association
(251). Among the remaining studies, the risk ratios reported for soy consumption and
colon or rectal cancer ranged from 0.48 to 1.9 (252-261). Only two studies found a

significant inverse association between soy consumption and colon cancer after

26

adjustment for multiple confounding variables. The first reported a 50% lower risk of
colon cancer only in women consuming higher soy products (260) and the other found a
50% reduction in risk of colorectal polyps with increased consumption of tofu or
soybeans (259). The interpretation of results from the remainder of the studies is limited
due to the incomplete assessment of soy intake and because most studies did not adjust
for confounding variables (248). However, despite these limitations, there was some
suggestion of an inverse association between soy consumption and colon cancer and
between non-fermented soy products and rectal cancer (248).

One clinical study was conducted to determine if consumption of soy protein
isolate could reduce early biomarkers associated with colon cancer development (262).
Subjects with a history of colon cancer or polyps consumed 38 g of soy protein isolate
containing 70 mg of isoflavones or 38 g of casein for one year. Biopsies of colon tissue
were taken at the start of the study and after one year of intervention. The author
reported a downward shift in the proliferation zone in colonic crypts obtained from
individuals consuming the soy protein isolate, suggesting soy consumption reduces colon

cancer risk by enhancing cellular differentiation (262).

2,; Experimental studies.

Only two studies have examined the potential of dry beans to inhibit colon cancer.
Hughes et al. (14) fed rats diets containing either pinto beans or casein as the protein
source and found that feeding pinto beans inhibited colon cancer by 50% and
significantly reduced the number of tumors that developed. In another study, Hangen and

Bennink (15) reported that feeding either black beans or navy beans inhibited colon

27

cancer by ~57%, and similar to Hughes et al. (14), bean-fed rats also developed fewer
tumors. In this study, the chemoprevention of beans was associated with significantly
more resistant starch reaching the colon, higher colonic acetate and butyrate production,
and a decrease in body fat (15).

The experimental data examining the potential of soy-based diets to inhibit
chemically induced colon cancer in rodents is inconsistent. This is due, in part to
differences in experimental protocols, the type of soy products (soy flour/meal or isolated
soy proteins) utilized, and end-point biomarkers examined. Processing of soybeans can
affect the content of carbohydrates, fiber, and various bioactive compounds. Because
some of these compounds have been reported to have anti-carcinogenic properties, it is
likely that consumption of soy products that are minimally processed may be more
beneficial in reducing risk of colon cancer.

Experiments where soy flour was examined and feeding began following
carcinogen administration (promotion), a protective effect on both preneoplastic lesions
and colon cancer has been reported (16-18, 263). In a series of studies, Bennink et al.
(16-18) compared the potential of diets containing soy flour (full-fat or defatted), casein,
or ethanol-washed soy concentrate to inhibit colonic tumor development during the
promotional phase of carcinogenesis. They consistently reported a reduction in colon
tumor incidence and tumor burden in rats fed defatted soy flour (43%, 0.67
tumors/animal) compared to rats fed either soy concentrate (68%, 1.1 tumor/animal) or
casein (68%, 1.43 tumor/animal). However, two animal studies that fed rats either soy
flour or soybean meal both prior to and after carcinogen administration have produced

inconsistent results (264-265). One found an increase in colon tumors (264) in soybean

28

meal-fed animals (P = 0.15) whereas the other study found a significant reduction in
tumor incidence in animals fed defatted soy flour (P < 0.05) (265).

Five experimental studies have examined the influence of soy protein isolates
high or low in isoflavones on colon carcinogenesis (44, 266-269). With the exception of
one study (269), isolates do not appear to influence colon tumorigenesis (44), and may
enhance the development of ACF when fed prior to carcinogen injection (44, 267-268),
although this does not appear to correlate with increased tumorigenesis (44). These
studies indicate that minimally processed soy products more consistently inhibit colon
cancer than either soy concentrates or isolates and chemoprevention appears associated

with one or more bioactive compounds present in soy.

IE Non-nutritive bioactive compounds.

The inverse relationship between consumption of plant-based foods and lowered
risk of several cancers has generated interest in identifying compounds with anti-cancer
potential. Legumes contain several compounds including saponins, protease (Bowman-
Birk) inhibitors, phenolics, phytoestrogens, and phytic acid that have been examined with
respect to colon cancer inhibition. In an attempt to summarize these studies, protease
inhibitors (270-272) and phytic acid (273-276) inhibit colon cancer most consistently in
animal models, whereas phytoestrogens (17, 277-279) do not appear to modulate colon
cancer risk. Limited data are available for saponins (280) and phytosterols (281).

The Bowman-Birk protease inhibitor (B31) and phytic acid (IPG) have been the
most extensively studied components in legumes, and both consistently inhibit
BXperimental carcinogenesis in the colon (270-276) and at other sites (282-289), The

mechanisms of inhibition by either compound are not known with certainty. BBIs are

29

small (6-10 kDa) proteins that can bind to and inhibit both chymotrypsin and trypsin
(143). BBI distributes among most tissues within 3 hours following oral administration,
the highest concentrations remaining in intestinal contents and urine (290). Because the
catalytic activity within tissues remains intact (291), Kennedy proposed that BBI may be
inhibiting one or more enzymes involved in inducing the transformed phenotype (148-
149, 291). In support of this, BBI has been associated with reversing the up-regulation of
proteolytic activity in the oral epithelium following carcinogen treatment (292) and
suppressing radiation-induced expression of proto-oncogenes (c-myc) in colon tissue and
cancer cell lines (170, 293).

Vucinek and Shamsuddin (294) suggested that phytic acid (IPg) inhibits tumor
growth by affecting cellular signaling through lower inositol derivatives. There is some
evidence that 1P6 interacts with the Akt-NFKB cell survival pathway (295). Sandra et al.
(295) reported that pre-treatment of 1P6 to HeLa cells inhibited insulin or TNF induced
Akt phosphorylation and NFKB translocation into the nucleus. In this study, neither PI-3
kinase activity nor the MAP kinase signaling pathway was affected by [P6, however there
was a significant reduction of Akt translocation to the plasma membrane, potentially
through antagonist binding of IR; to the pleckstrin homology domain of Akt (295).
Treatment of cells with IP6 induces a G1 cell cycle arrest and reduces cells in the S phase
in breast (296), colon (296), and prostate cancer cell lines (297). Additionally, 1P6 and
inositol derivatives containing at least 3 phosphates (1P5, [P4, and 1P3) can bind divalent
metals, and reduces iron catalyzed lipid peroxidation (298) and high iron-induced

promotion of colon tumorigenesis in rats (299).

30

Most of the experimental evidence suggesting phytoestrogens, in particular
genistein, may prevent cancer has been derived from in vitro studies. Genistein
suppresses growth and induces apoptosis in a variety of cancer cell lines (300-308).
Genistein has also been shown to inhibit lipid oxidation (309-310), suppress angiogenesis
(311-314), and at high concentrations inhibits tyrosine kinases (315). However, animal
studies examining the potential of purified phytoestrogens to modulate colon
carcinogenesis are generally not supportive of a protective effect (17, 277-279). Four
studies have been conducted long enough for tumors to develop and neither found a
significant reduction in colon tumorigenesis by feeding genistein (277, 279), genistin
(17), or a mixture of soy isoflavones (17, 280-281). In one study, genistein (250 ug/g
diet) enhanced tumor multiplicity in the colon of rats administered AOM (277).
Similarly, Bennink et al. (17) fed genistin or a mixture of isoflavones (genistin, daidzin,
and glycitin) in amounts comparable to that in defatted soy flour and found that genistin
increased the number of rats with tumors (P = 0.06), although the tumors that developed
were smaller than in controls. The other two studies found no effect of phytoestrogens on
intestinal tumorigenesis (278-279). Therefore, phytoestrogens fed as purified compounds

are likely not sufficient to inhibit colon cancer in established animal models.

31

E. RATIONALE.

CRC is a significant health problem in industrialized countries. In the United
States, it is the fourth most common cancer and the second leading cause of cancer
mortality (19). Despite improvements in adjunct therapy for those diagnosed with
colorectal cancer, the relative five-year survival rate is currently only 63% (19). When
detected at an early, localized stage, the overall prognosis improves. However, due to
low rates of screening in the general population, only 39% of CRC cases are diagnosed at
this early stage (19). Clearly, prevention strategies aimed at early detection and
modifying risk factors have the potential to reduce medical costs and age-adjusted
mortality associated with the disease.

Dietary habits strongly influence the development of CRC, and dietary factors
associated with an increased or decreased risk for CRC have been identified (2-4)
(TABLE 1). Several of the mechanisms proposed to explain the relationship of diet to
risk of colon tumorigenesis are related to modulation of mucosal abnormalities that favor
adenoma formation. For example, in the colon of individuals at high risk for CRC and in
animals treated with colon carcinogens, an upward shifi of proliferative epithelial cells
towards the lumen has been described (37, 40). The anti-cancer properties of some
dietary components have been attributed to reversing mucosal hyperproliferation (124-
129, 262), either through enhancing apoptotic indices (200, 202), favoring differentiation
(200, 202, 262), and/or modulating mitogenic enzyme activity (201, 205-208).

Recent studies have begun to explore dietary differences in mucosal gene
expression utilizing microarrays, and have revealed a more complex involvement of diet

on expression of genes involved in apoptosis, immunity, and endocrine changes

32

potentially contributing to colon cancer inhibition (316-317). Because the anti-neoplastic
effects of many dietary components precede adenoma formation, further characterization
of these events may lead to a more thorough understanding of diet on CRC etiology and
provide a rationale basis for specific public health recommendations aimed at colon
cancer prevention.

The AOM-induced rodent model is frequently utilized to examine dietary
influences on colon carcinogenesis. Using this model, dry beans (14-15) and soy flour
(16-18) have been previously demonstrated to inhibit the both the incidence and
multiplicity of colonic tumors compared to rats fed a control (casein-based) diet. The
mechanisms underlying altered susceptibility to colon cancer by beans have not been
fully elucidated. Because beans inhibit the occurrence and the number of tumors (14-18)
that develop rather than the size of colon tumors (mg tumor/tumor bearing rat) (15), the
current study was designed to examine potential chemopreventative mechanisms of beans
early in the neoplastic process. Therefore, the central hypothesis of this research is that
beans inhibit colon cancer by modulating mucosal expression of genes regulating
epithelial cell grth and differentiation. The primafl objectives were to: 1) identify
genes involved in cellular grth and differentiation that are significantly increased or
decreased in colon tumors compared to normal colonic epithelium; and 2) identify which
of these gene changes in non-neoplastic mucosa were attenuated by feeding beans using

microarrays.

To test my hypothesis, three experiments were conducted:

1. In the first experiment, microarrays were used to profile genes differentially

33

expressed in rat AOM-induced colon cancers compared to non-neoplastic colonic
mucosa. Because AOM is a global methylating agent and early gene changes have
not been previously described, results from this experiment provided insight into
biological processes involved in tumor promotion (CHAPTER III).

2. Having determined genetic alterations associated with cancer promotion, the second
experiment was conducted to profile genes altered in non-neoplastic mucosa to
identify early cellular events permissive for tumorigenesis and those affected by
dietary treatment. Microarrays were performed on RNA isolated from normal-
appearing colonic mucosa in rats treated with either carcinogen (AOM) or saline and
fed a control (casein)-, black bean-, or soy-flour based diet. I anticipated that genes
important in dietary suppression of colon carcinogenesis would have altered
expression (increased or decreased) that correlated with tumor incidence and

proceeded in the following sequence (CHAPTER IV).

 

 

A Colon tumors
Increased Colonic mucosa from Casein-fed, AOM-injected Decreased
mRNA c1' fr BlkBan/S fdAOM" d mRNA
expression 0 onrc mucosa om ac e oy- e , -1njecte expression
Colonic mucosa from Casein-fed, saline-injected
Colonic mucosa from Black Bean/Soy-fed, saline-injected i

3. The third experiment was conducted to determine if diet-related differences in gene
changes correspond to alterations in epithelial kinetics and colonic inflammation

using immunohistochemical techniques (CHAPTER V).

34

10.

ll.

12.

13.

F. LITERATURE CITED.

Ferlay J, Bray F, Pisani P and Parkin DM. (2002) GLOBOCAN 2002: Cancer
incidence, mortality, and prevalence worldwide. IARC Press, Lyon, France,
http://www-deglb.iarc.fr/globocan/GLOBOframehtm.

American Institute of Cancer Research/World Cancer Research Fund
(AICR/WCRF). (1997) Colon, rectum. In Food, Nutrition and the Prevention of
Cancer: A Global Perspective. BANT A Book Group, Menasha, WI, pp. 216-51.

Potter JD, Slattery ML, Bostick RM and Gapstur SM. (1993) Colon-Cancer - a
Review of the Epidemiology. Epidemiologic Reviews. 15(2): 499-545.

Potter JD. (1999) Colorectal cancer: Molecules and populations. Journal of the
National Cancer Institute. 91(11): 916-932.

Singh PN and Fraser GE. (1998) Dietary risk factors for colon cancer in a low-risk
population. American Journal of Epidemiology. 148: 761-774

Fraser GE. (1999) Associations between diet and cancer, ischemic heart disease,
and all-cause mortality in non-Hispanic white California Seventh-day Adventists.
American Journal of Clinical Nutrition. 70: 5328-5388.

Correa P. (1981) Epidemiological correlations between diet and cancer frequency.
Cancer Research. 41: 3685-3689

Vogelstein B, Fearon ER, Hamilton SR et al. (1988) Genetic Alterations During
Colorectal-Tumor Development. New England Journal of Medicine. 319(9): 525-
532.

Fearon ER and Vogelstein B. (1990) A Genetic Model for Colorectal
Tumorigenesis. Cell. 61(5): 759-767.

Kinzler KW and Vogelstein B. (1996) Lessons from hereditary colorectal cancer.
Cell. 87(2): 159-170.

Ilyas M, Straub J, Tomlinson [PM and Bodmer WF. (1999) Genetic pathways in
colorectal and other cancers. European Journal of Cancer. 35(3): 335-351.

Grady WM and Markowitz SD. (2002) Genetic and epigenetic alterations in colon
cancer. Annual Review of Genomics and Human Genetics. 3: 101-128.

Pories SE, Ramchurren N, Summerhayes I and Steele, G. (1993) Animal models
for colon carcinogenesis. Archives of Surgery. 128: 647-53.

35

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

Hughes JS, Ganthavom C and Wilson-Sanders S. (1997) Dry beans inhibit
azoxymethane-induced colon carcinogenesis in F344 rats. Journal of Nutrition.
127: 2328-2333

Hangen LA and Bennink MR. (2003) Consumption of black beans and navy beans
(Phaseolus vulgaris) reduced axozymethane-induced colon cancer in rats. Nutrition
and Cancer. 44: 60-6

Bennink MR and Om AS. (1998) Inhibition of colon cancer (CC) by soy
phytochemicals but not by soy protein. F aseb Journal. 12(5): 3808.

Bennink MR, Om AS and Miyagi Y. (1999) Inhibition of colon cancer (CC) by soy
flour but not by genistin or a mixture of isoflavones. Faseb Journal. 13(4): A50-
A50.

Bennink MR, Om AS and Miyagi Y. (2000) Inhibition of colon cancer (CC) by
wheat bran, soy flour, and flax. Faseb Journal. 14(4): A217-A217.

American Cancer Society. (2005) Cancer Facts & Figures 2005.
http://www.cancer.org/downloads/STT/CAFF2005f4PWSecured.pdf.

Troisi RJ, Freedman AN and Devesa SS. (1999) Incidence of colorectal carcinoma
in the US - An update of trends by gender, race, age, subsite, and stage, 1975-1994.
Cancer. 85(8): 1670-1676.

Calvert PM and Frucht H. (2002) The genetics of colorectal cancer. Annals of
Internal Medicine. 137(7): 603-612.

Bianchini F, Kaaks R, and Vainio H. (2002) Overweight, obesity, and cancer risk.
Lancet Oncology. 3(9): 565-574.

Calle EE, Rodriguez C, Walker-Thurmond K and Thun MJ. (2003) Overweight,
obesity, and mortality from cancer in a prospectively studied cohort of US adults.
New England Journal of Medicine. 348(17): 1625-1638.

Calle EE and Thun MJ. (2004) Obesity and cancer. Oncogene. 23(38): 6365-6378.

Jee SH, Kim H], and Lee J. (2005) Obesity, insulin resistance and cancer risk.
Yonsei Medical Journal. 46(4): 449-455.

Komninou D, Ayonote A, Richie JP and Rigas B. (2003) Insulin resistance and its
contribution to colon carcinogenesis. Experimental Biology and Medicine. 228(4):
396-405.

Giovannucci E. (2003) Nutrition, insulin, insulin-like grth factors and cancer.
Hormone and Metabolic Research. 35(1 1-12): 694-704.

36

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

Chang CK and Ulrich CM. (2003) Hyperinsulinaemia and hyperglycaemia:
possible risk factors of colorectal cancer among diabetic patients. Diabetologia.
46(5): 595-607.

Eaden J. (2004) Review article: colorectal carcinoma and inflammatory bowel
disease. Alimentary Pharmacology & Therapeutics. 20: 24-30.

Samad AKA, Taylor RS, Marshall T and Chapman MAS. (2005) A meta-analysis
of the association of physical activity with reduced risk of colorectal cancer.
Colorectal Disease. 7(3): 204-21 3.

Herendeen JM and Lindley C. (2003) Use of NSAIDs for the chemoprevention of
colorectal cancer. Annals of Pharmacotherapy. 37(11): 1664-1674.

Grodstein F, Newcomb PA and Stampfer MJ. (1999) Postmenopausal hormone

therapy and the risk of colorectal cancer: A review and meta-analysis, American
Journal of Medicine. 106: 574-82.

Cummings JH and Macfarlane GT. (1991) The control and consequences of

bacterial fermentation in the human colon. Journal of Applied Bacteriology. 70:
443-59.

Eastwood GL. (1983) Colon structure. In Colon, structure and function, (Bustos-
Fernandez L., ed.), Plenum Medical Book Company, New York, pp. 1-16.

Kinzler KW and Vogelstein B. (1998) Colorectal tumors. In The genetic basis of

human cancer, (Vogelstein B and Kinzler KW, eds.), McGraw Hill, New York, pp.
$65-82.

Karam SM. (1999) Lineage commitment and maturation of epithelial cells in the
gut. Frontiers in Bioscience. 4: d286-98.

Lipkin M. (1974) Phase I and phase II proliferative lesions of colonic epithelial
cells in diseases leading to colonic cancer. Cancer. 34: 878-888.

Muto T, Bussey JR and Morson BC. (1975) The evolution of cancer of the colon
and rectum. Cancer. 36: 2251-70.

Fenoglio CM and Lane N. (1974) The anatomical precursor of colorectal
carcinoma. Cancer. 34: 819-23.

Winawer SJ, Zauber AG, Steward E et al. (1991) The natural history of colorectal
cancer: opportunities for intervention. Cancer. 67: 1143-49.

Bird RP. (1995) Role of aberrant crypt foci in understanding the pathogenesis of
colon cancer. Cancer Letters. 93: 55-71.

37

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

Siu 1M, Pretlow TG, Amini SB and Pretlow TP. (1997) Identification of dysplasia

in human colonic aberrant crypt foci. American Journal of Pathology. 150(5):
1805-1813.

Hardman WE, Cameron IL, Heitrnan DW and Contreras E. (1991) Demonstration
of the Need for End-Point Validation of Putative Biomarkers - Failure of Aberrant
Crypt Foci to Predict Colon Cancer Incidence. Cancer Research. 51(23): 6388-
6392.

Davies MJ, Bowey EA, Adlercreutz H et al. (1999) Effects of soy or rye
supplementation of high-fat diets on colon tumour development in azoxymethane-
treated rats. Carcinogenesis. 20(6): 927-931.

Oving IM and Clevers HC. (2002) Molecular causes of colon cancer. European
Journal of Clinical Investigation. 32(6): 448-457.

Vogelstein B and Kinzler KW. (1993) The Multistep Nature of Cancer. Trends in
Genetics. 9(4): 138-141.

Powell SM, Zilz N, Beazer Barclay Y et al. (1992) APC mutations occur early
during colorectal tumorigenesis. Nature. 359: 235-37.

Miyaki M, Konishi M, Kikuchiyanoshita R et al. (1994) Characteristics of Somatic
Mutation of the Adenomatous Polyposis-Coli Gene in Colorectal Tumors. Cancer
Research. 54(11): 3011-3020.

Polakis P. (1997) The adenomatous polyposis coli (APC) tumor suppressor.
Biochimica Et Biophysica Acta-Reviews on Cancer. 1332(3): F 127-Fl47.

van Es JH, Giles RH and Clevers HC. (2001) The many faces of the tumor
suppressor gene APC. Experimental Cell Research. 264(1): 126-134.

Kuhl M and Wedlich D. (1997) Wnt signalling goes nuclear. Bioessays. 19(2): 101-
104.

Morin PJ. (1999) Beta-catenin signaling and cancer. Bioessays. 21(12): 1021-1030.
Orford K, Crockett C, Jensen JP et al. (1997) Serine phosphorylation-regulated
ubiquitination and degradation of beta-catenin. Journal of Biological Chemistry.
272(40): 24735-24738.

Hedgepeth CM, Deardorff MA, Rankin K and Klein PS. (1999) Regulation of

glycogen synthase kinase 3 beta and downstream Wnt signaling by axin. Molecular
and Cellular Biology. 19(10): 7147-7157.

38

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

65.

66.

Yokoya F, Irnamoto N, Tachibana T and Yoneda Y. (1999) Beta-catenin can be
transported into the nucleus in a Ran-unassisted manner. Molecular Biology of the
Cell. 10(4): 1119-1131.

Chung DC. (2000) The genetic basis of colorectal cancer: insights into critical
pathways of tumori genesis. Gastroenterology. 119: 854-65.

He TC, Sparks AB, Rago C et al. (1998) Identification of c-MYC as a target of the
APC pathway. Science. 281(5382): 1509-1512.

Shtutrnan M, Zhurinsky J, Simcha I et al. (1999) The cyclin D1 gene is a target of
the beta-catenin/LEF-l pathway. Proceedings of the National Academy of Sciences
of the United States of America. 96(10): 5522-5527.

Mann B, Gelos M, Siedow A et al. (1999) Target genes of beta-catenin-T cell-
factor lymphoid-enhancer-factor signaling in human colorectal carcinomas.
Proceedings of the National Academy of Sciences of the United States of America.
96(4): 1603-1608.

Zhang T, Otevrel T, Gao ZQ et al. (2001) Evidence that APC regulates survivin
expression: A possible mechanism contributing to the stem cell origin of colon
cancer. Cancer Research. 61(24): 8664-8667.

Brabletz T, Jung A, Dag S et al. (1999) Beta-catenin regulates the expression of the
matrix metalloproteinase-7 in human colorectal cancer. American Journal of
Pathology. 155(4): 1033-1038.

Gradl D, Kuhl M and Wedlich D. (1999) The Wnt/W g signal transducer beta-
catenin controls fibronectin expression. Molecular and Cellular Biology. 19(8):
5576-5587.

He TC, Chan TA, Vogelstein B and Kinzler KW. (1999) PPAR delta is an APC-
regulated target of nonsteroidal anti-inflammatory drugs. Cell. 99(3): 335-345.

Sparks AB, Morin PJ, Vogelstein B and Kinzler KW. (1998) Mutational analysis of
the APC/beta-catenin/T cf pathway in colorectal cancer. Cancer Research. 58(6):
1130-1134.

Vojtek AB and Der CJ. (1998) Increasing complexity of the Ras signaling pathway.
Journal of Biological Chemistry. 273(32): 19925-19928.

Hedrick L, Cho KR, Fearon ER et al. (1994) The Doc Gene-Product in Cellular-

Differentiation and Colorectal Tumori genesis. Genes & Development. 8(10): 1174-
1183.

39

67.

68.

69.

70.

71.

72.

73.

74.

75.

76.

77.

78.

79.

Massague J. (2000) How cells read TGF-beta signals. Nature Reviews Molecular
Cell Biology. 1(3): 169-178.

Fridman J S and Lowe SW. (2003) Control of apoptosis by p53. Oncogene. 22(56):
9030-9040.

Taylor WR and Stark GR. (2001) Regulation of the GZ/M transition by p53.
Oncogene. 20: 1803-15.

Wang XW, Zhan Q, Coursen JD et al. (1999) Gadd induction of a G2/M cell cycle
check point. Proceedings of the National Academy of Sciences, USA. 96: 3706-11.

Noda A, Ning Y, Venable SF et al. (1994) Cloning of Senescent Cell-Derived
Inhibitors of DNA-Synthesis Using an Expression Screen. Experimental Cell
Research. 21 1(1): 90-98.

Eldeiry WS, Tokino T, Velculescu VE et al. (1993) Wafl, a Potential Mediator of
P53 Tumor Suppression. Cell. 75(4): 817-825.

Kastan MB, Zhan QM, Eldeiry WS et al. (1992) A Mammalian-Cell Cycle
Checkpoint Pathway Utilizing P53 and Gadd45 Is Defective in Ataxia-
Tclangiectasia. Cell. 71(4): 587-597.

Kondo Y and Issa JPJ. (2004) Epigenetic changes in colorectal cancer. Cancer and
Metastasis Reviews. 23(1): 29-39.

Goelz SE, Vogelstein B, Hamilton SR, and Feinberg AP. (1985) Hypomethylation
of DNA from benign and malignant human colon neoplasms. Science. 228(4696):
187-190.

Waddell WR and Loughry RW. (1983) Sulindac for Polyposis of the Colon.
Journal of Surgical Oncology. 24(1): 83-87.

Herendeen J M and Lindley C. (2003) Use of NSAIDs for the chemoprevention of
colorectal cancer. Annals of Pharmacotherapy. 37(11): 1664-1674.

Brown WA, Skinner SA, Malcontenti-Wilson C et al. (2001) Non-steroidal anti-
inflammatory drugs with activity against either cyclooxygenase 1 or
cyclooxygenase 2 inhibit colorectal cancer in a DMH rodent model by inducing
apoptosis and inhibiting cell proliferation. Gut. 48(5): 660-666.

Jacoby RF, Seibert K, Cole CE et al. (2000) The cyclooxygenase-2 inhibitor

celecoxib is a potent preventive and therapeutic agent in the min mouse model of
adenomatous polyposis. Cancer Research. 60(18): 5040-5044.

40

80.

81.

82.

83.

84.

85.

86.

87.

88.

89.

90.

Yoshimi N, Shimizu M, Matsunaga K et al. (1999) Chemopreventive effect of N-
(2-cyclohexyloxy-4-nitrophenyl)methane sulfonamide (NS-398), a selective
cyclooxygenase-2 inhibitor, in rat colon carcinogenesis induced by azoxymethane.
Japanese Journal of Cancer Research. 90(4): 406-412.

Ritland SR and Gendler SJ. (1999) Chemoprevention of intestinal adenomas in the
Apc(Min) mouse by piroxicam: kinetics, strain effects and resistance to
chemosuppression. Carcinogenesis. 20(1): 51-58.

Reddy BS, Rao CV and Seibert K. (1996) Evaluation of cyclooxygenase-2 inhibitor
for potential Chemopreventive properties in colon carcinogenesis. Cancer Research.
56(20): 4566-4569.

Reddy BS, Hirose Y, Lubet R et al. (2000) Chemoprevention of colon cancer by
specific cyclooxygenase-2 inhibitor, celecoxib, administered during different stages
of carcinogenesis. Cancer Research. 60(2): 293-297.

Boolbol SK, Dannenberg AJ, Chadbum A et al. (1996) Cyclooxygenase-2
overexpression and tumor formation are blocked by sulindac in a murine model of
familial adenomatous polyposis. Cancer Research. 56(11): 2556-2560.

Huls G, Koornstra JJ and Kleibeuker JH. (2003) Non-steroidal anti-inflammatory

drugs and molecular carcinogenesis of colorectal carcinomas. Lancet. 362(9379):
230-232.

Ashktorab H, Dawkins FW, Mohamed R et al. (2005) Apoptosis induced by aspirin
and 5-fluorouracil in human colonic adenocarcinoma cells. Digestive Diseases and
Sciences. 50(6): 1025-1032.

Pelzmann M, Thumher D, Gedlicka C et al. (2004) Nimesulide and indomethacin
induce apoptosis in head and neck cancer cells. Journal of Oral Pathology &
Medicine. 33(10): 607-613.

Tarnawski AS and Jones MK. (2003) Inhibition of angiogenesis by NSAIDs:
molecular mechanisms and clinical implications. Journal of Molecular Medicine-
Jmm. 81(10): 627-636.

Cheong E, Ivory K, Doleman J et al. (2004) Synthetic and naturally occurring
COX-2 inhibitors suppress proliferation in a human oesophageal adenocarcinoma

cell line (OE33) by inducing apoptosis and cell cycle arrest. Carcinogenesis.
25(10): 1945-1952.

DuBois RN, Radhika A, Reddy BS and Entingh AJ. (1996) Increased

cyclooxygenase-2 levels in carcinogen-induced rat colonic tumors.
Gastroenterology. 1 10(4): 125 9-1 262.

41

91.

92.

93.

94.

95.

96.

97.

98.

99.

100.

101.

102.

Oshima M, Dinchuk JE, Kargman SL et al. (1996) Suppression of intestinal
polyposis in Apc(Delta 716) knockout mice by inhibition of cyclooxygenase 2
(COX-2). Cell. 87(5): 803-809.

Piazza GA, Alberts DS, Hixson LJ et al. (1997) Sulindac sulfone inhibits
azoxymethane-induced colon carcinogenesis in rats without reducing prostaglandin
levels. Cancer Research. 57(14): 2909-2915.

Hanif R, Pittas A, Feng Y et a1. (1996) Effects of nonsteroidal anti-inflammatory
drugs on proliferation and on induction of apoptosis in colon cancer cells by a
prostaglandin-independent pathway. Biochemical Pharmacology. 52(2): 237-245.

Hanif R, Pittas A, F eng Y, et al. (1995) Nsaids Inhibit the Growth of Colon-Cancer
Cell-Lines by a Prostaglandin Independent Pathway. Gastroenterology. 108(4):
A478-A478.

Druckrey H, Preussman R, Matzkies F and Ivankovic S. (1967) Erzeugung von
Darmkrebs bei Ratten durch 1,2-dimethylhydrazin. Naturewissenschaften. 54: 285-
286.

Druckrey H. (1968) Organotropic carcinogenesis and mechanisms of action of
symmetrical dialkylhydrazines and azo-and azoalkanes. Food Cosmet. T oxicol. 6:
578-579.

Druckery H. (1970) Production of colonic carcinomas by 1,2-dialkylhydrazines and
azoalkanes. In Carcinoma of the colon and antecedent epithelium, (WJ Burdette,
ed.), Charles C Thomas, Springfield, IL, pp. 267-79.

Hong MY, Chapkin RS, Wild CP et al. (1999) Relationship between DNA adduct
levels, repair enzyme, and apoptosis as a function of DNA methylation by
azoxymethane. Cell Growth & Differentiation. 10(11): 749-758.

Hollstein M, Sidransky D, Vogelstein B and Harris CC. (1991) P53 Mutations in
Human Cancers. Science. 253(5015): 49-53.

Takahashi M, Fukuda K, Sugimura T and Wakabayashi K. (1998) Beta-catenin is
frequently mutated and demonstrates altered cellular location in azoxymethane-
induced rat colon tumors. Cancer Research. 58(1): 42-46.

Jacoby RF, Llor X, Teng BB et al. (1991) Mutations in the K-Ras Oncogene
Induced by 1,2-Dimethylhydrazine in Preneoplastic and Neoplastic Rat Colonic
Mucosa. Journal of Clinical Investigation. 87(2): 624-630.

Vivona AA, Shpitz B, Medline A et al. (1993) K-Ras Mutations in Aberrant Crypt

Foci, Adenomas and Adenocarcinomas During Azoxymethane-Induced Colon
Carcinogenesis. Carcinogenesis. 14(9): 1777-1781.

42

103.

104.

105.

106.

107.

108.

109.

110.

111.

112.

113.

114.

Takahashi M and Wakabayashi K. (2004) Gene mutations and altered gene
expression in azoxymethane-induced colon carcinogenesis in rodents. Cancer
Science. 95(6): 475-480.

Watanabe K, Kawamori T, Nakatsugi S and Wakabayashi K. (2000) COX-2 and
iNOS, good targets for chemoprevention of colon cancer. Biofactors. 12(1-4): 129-
133.

Sandler RS. (1996) Epidemiology and risk factors for colorectal cancer.
Gastroenterol Clin North America. 25(4): 717-35.

McKeown-Eyssen G. (1994) Epidemiology of colorectal cancer revisited: are
serum triglycerides and/or plasma glucose associated with risk?
Cancer Epidemiology Biomarkers & Prevention. 3(8): 687-95.

Giovannucci E. (1995) Insulin and Colon-Cancer. Cancer Causes & Control. 6(2):
164-179.

Bianchini F, Kaaks R and Vainio H. (2002) Overweight, obesity, and cancer risk.
Lancet Oncology. 3(9): 565-574.

Chang CK and Ulrich CM. (2003) Hyperinsulinaemia and hyperglycaemia:
possible risk factors of colorectal cancer among diabetic patients. Diabetologia.
46(5): 595-607.

Komninou D, Ayonote A, Richie JP and Rigas B. (2003) Insulin resistance and its
contribution to colon carcinogenesis. Experimental Biology and Medicine. 228(4):
396-405.

Meyerkardt JA, Catalano PJ, Haller DG et al. (2003) Impact of diabetes mellitus on
outcomes in patients with colon cancer. Journal of Clinical Oncology. 21(3): 433-
440.

Kaaks R, Toniolo P, Akhmedkhanov A et al. (2000) Serum C-peptide, insulin-like
grth factor (IGF)-I, IGF-binding proteins, and colorectal cancer risk in women.
Journal of the National Cancer Institute. 92(19): 1592-1600.

Cross AJ and Sinha R. (2004) Meat-related mutagens/carcinogens in the etiology of
colorectal cancer. Environmental and Molecular Mutagenesis. 44(1): 44-55.

Norat T, Lukanova A, Ferrari P and Riboli E. (2002) Meat consumption and

colorectal cancer risk: Dose-response meta-analysis of epidemiological studies.
International Journal of Cancer. 98(2): 241-256.

43

115.

116.

117.

118.

119.

120.

121.

122.

123.

124.

125.

Malfatti MA, Shen NH, Wu RW et al. (1995) A Correlation of Salmonella
Mutagenicity with DNA-Adducts Induced by the Cooked-Food Mutagen 2-Amino-
1-Methyl-6-Phenylimidazo[4,5-B]Pyridine. Mutagenesis. 10(5): 425-43 1 .

Ohgaki H, Takayama S and Sugimura T. (1991) Carcinogenicities of Heterocyclic
Amines in Cooked Food. Mutation Research. 259(3-4): 399-410.

Hasegawa R, Sano M, Tamano S et al. (1993) Dose-Dependence of 2-Amino-1-
Methyl-6-Phenylimidazo[4,5-B]-Pyridine (Phip) Carcinogenicity in Rats.
Carcinogenesis. 14(12): 2553-2557.

Reddy BS. (1986) Diet and colon cancer: evidence from human and animal model
studies. (Reddy B. S. Cohen L. A. eds.). In Diet, Nutrition, and Cancer: A Critical
Evaluation, CRC Press Boca Raton, FL , pp. 47-65.

Hill MJ, Crowther J S, Drasar BS et al. (1971) Bacteria and the aetiology of cancer
in the large bowel. Lancet. 1: 95-100.

Velaquez OC, Zhou D, Seto RW et al. (1996) In vivo crypt surface
hyperproliferation is decreased by butyrate and increased by deoxycholate in

normal rat colon: associated in vivo effects on c-Fos and c-Jun expression. J.P.E.N.
20: 243-50.

Narisawa T, Magadia NE, Weinsburger JH and Wynder EL. (1974) Promoting
effect of bile acids on colon carcinogenesis afier intrarectal instillation of N-
methyl-N-nitro-N-nitrosoguanidine in rats. JNCI. 53: 1093-97.

Reddy BS, Watanabe K, Weisburger JH and Wynder EL. (1977) Promoting effect
of bile acids in colon carcinogenesis in germ free and conventional F344 rats.
Cancer Research. 37: 3238-42.

Summerton J, Goeting N, Trotter GA and Taylor I. (1985) Effect of deoxycholic
acid on the tumor incidence, distribution, and receptor status of colorectal cancer in
the rat model. Digestion. 31: 77-81.

Beaty MM, Lee EY and Glauert HP. (1993) Influence of Dietary Calcium and
Vitamin-D on Colon Epithelial-Cell Proliferation and 1,2-Dimethylhydrazine-
Induced Colon Carcinogenesis in Rats Fed High-Fat Diets. Journal of Nutrition.
123(1): 144-152.

Lapre JA, Devries HT, Koeman JH and Vanderrneer R. (1993) The
Antiproliferative Effect of Dietary Calcium on Colonic Epithelium Is Mediated by
Lumenal Surfactants and Dependent on the Type of Dietary-Fat. Cancer Research.
53(4): 784-789.

44

126.

127.

128.

129.

130.

131.

132.

133.

134.

135.

136.

137.

138.

Lipkin M and Newmark H. (1985) Effect of added dietary calcium on colonic
epithelial cell proliferation in subjects at high risk for familial colonic cancer. New
England Journal of Medicine. 313: 1381-1384.

Rozen, PA, Fireman N, Fine et al. (1989) Oral calcium suppresses increased rectal
epithelial proliferation of persons at risk for colorectal cancer. Gut. 30: 650-655.

Wargovich, MJ, Isbell G, Shabot M et al. (1992) Calcium supplementation
decreases rectal epithelial cell proliferation in subjects with sporadic adenoma.
Gastroenterology. 103: 92-97.

Holt PR, Wolper C, Moss SF et al. (2001) Comparison of calcium supplementation
or low-fat dairy foods on epithelial cell proliferation and differentiation. Nutrition
and Cancer-an International Journal. 41(1-2): 150-155.

Simanowski UA, Homann N, Knuhl M et al. (2001) Increased rectal cell
proliferation following alcohol abuse. Gut. 49(3): 418-422.

Simanowski UA, Suter P, Russell RM et al. (1994) Enhancement of Ethanol-
Induced Rectal Mucosal Hyper Regeneration with Age in F344 Rats. Gut. 35(8):
1 102-1 106.

Heavey PM, McKenna D and Rowland IR. (2004) Colorectal cancer and the
relationship between genes and the environment. Nutrition and Cancer-an
International Journal. 48(2): 124-141.

Giovannucci E, Rimm EB, Ascherio A et al. (1995) Alcohol, low methionine-low
folate diets, and risk for colon cancer in men. J Natl Cancer Int. 87:265-273.

Su LJ and Arab L. (2001) Nutritional status of folate and colon cancer risk:
Evidence from NHANES I Epidemiologic Follow-up Study. Annals of
Epidemiology. 11(1): 65-72.

Groff JL and Gropper SS. (2000) Folic Acid. In Advanced Nutrition and Human
Metabolism, 3rd Edition, West Publishing Company, St. Paul, MN, pp. 262-270.

Mason J and Choi SW. (2000) Folate and carcinogenesis: Developing a unifying
hypothesis. Advan Enzyme Regul. 40:127-141.

Choi SW and Mason JB. (2002) Folate status: Effects on pathways of colorectal
carcinogenesis. Journal of Nutrition. 132(8): 2413S-2418S.

Hoffman RM. (1984) Altered methionine metabolism, DNA methylation and

oncongene expression in carcinogenesis: A review and synthesis. Biochim Biophys
Acta. 738:49-87.

45

139.

140.

141.

142.

143.

144.

145.

146.

147.

148.

149.

150.

Kim YI, Shirwadkar S, Choi SW et al. (2000) Effects of dietary folate on DNA
strand breaks within mutation-prone exons of the p53 gene in rat colon.
Gastroenterology. 119(1): 151-161.

Slattery ML, Curtin K, Anderson K et al. (2000) Associations between dietary
intake and Ki-ras mutations in colon tumors: A population-based study. Cancer
Research. 60(24): 6935-6941.

Martinez ME, Maltzman T, Marshall JR et al. (1999) Risk factors for Ki-ras
protooncogene mutation in sporadic colorectal adenomas. Cancer Research.
59(20): 5181-5185.

Steinmetz KA and Potter JD. (1991) Vegetables, fruit, and cancer. 11. Mechanisms.
Cancer, Causes, and Control. 2: 427-442.

Kennedy AR. (1993) Cancer Prevention by Protease Inhibitors. Preventive
Medicine. 22(5): 796-81 1.

Kennedy AR. (1994) Prevention of Carcinogenesis by Protease Inhibitors. Cancer
Research. 54(7): S1999-S2005.

Reddy BS and Rao CV. (1994) Chemoprevention of Colon-Cancer by Thiol and
Other Organosulfur Compounds. Food Phytochemicals for Cancer Prevention 1.
546: 164-172.

Smith T, Musk SR and Johnson IT. (1996) Ally] isothiocyanate selectively kills
undifferentiated HT29 cells in vitro and suppresses aberrant crypt foci in the
colonic mucosa of rats. Biochem. Soc. Trans. 24: 3818.

Rijnkels J M, Hollanders VMH, Woutersen RA et al. (1997) Modulation of dietary
fat-enhanced colorectal carcinogenesis in N-methyl-N'-nitro-N-nitrosoguanidine-
treated rats by a vegetables-fiuit mixture. Nutrition and Cancer-an International
Journal. 29(1): 90-95.

Kennedy AR. (1998) The Bowman-Birk inhibitor from soybeans as an
anticarcinogenic agent. American Journal of Clinical Nutrition. 68(6): 1406S-
1412S.

Kennedy AR. (1998) Chemopreventive agents: Protease inhibitors. Pharmacology
& Therapeutics. 78(3): 167-209.

Chung FL, Conaway CC, Rao CV and Reddy BS. (2000) Chemoprevention of

colonic aberrant crypt foci in Fischer rats by sulforaphane and
phenethylisothiocyanate. Carcinogenesis. 2 1(12): 2287-2291 .

46

151.

152.

153.

154.

155.

156.

157.

158.

159.

160.

161.

162.

Seow A, Yuan JM, Sun CL et al. (2002) Dietary isothiocyanates, glutathione S-
transferase polymorphisms and colorectal cancer risk in the Singapore Chinese
Health Study. Carcinogenesis. 23(12): 2055-2061.

Emerit I. (1994) Reactive Oxygen Species, Chromosome Mutation, and Cancer -
Possible Role of Clastogenic Factors in Carcinogenesis. Free Radical Biology and
Medicine. 16(1): 99-109.

Gate L, Paul J, Ba GN et al. (1999) Oxidative stress induced in pathologies: the
role of antioxidants. Biomedicine & Pharmacotherapy. 53(4): 169-180.

Zhu CY, Poulsen HE and Loft S. (2000) Inhibition of oxidative DNA damage in
vitro by extracts of Brussels sprouts. Free Radical Research. 33(2): 187-196.

Feng Q, Kumagai T, Torii Y et al. (2001) Anticarcinogenic antioxidants as
inhibitors against intracellular oxidative stress. Free Radical Research. 35(6): 779-
788.

Bartsch H, Nair J and Owen RW. (2002) Exocyclic DNA adducts as oxidative
stress markers in colon carcinogenesis: Potential role of lipid peroxidation, dietary
fat and antioxidants. Biological Chemistry. 383(6): 915-921.

Flagg EW, Coates RJ and Greenberg RS. (1995) Epidemiologic Studies of
Antioxidants and Cancer in Humans. Journal of the American College of Nutrition.
14(5): 419-427.

Neugut AI, Horvath K, Whelan RL et al. (1996) The effect of calcium and vitamin
supplements on the incidence and recurrence of colorectal adenomatous polyps.
Cancer. 78(4): 723-728.

Jacobs EJ, Connell CJ, Patel AV et a1. (2001) Vitamin C and vitamin E supplement
use and colorectal cancer mortality in a large American Cancer Society cohort.
Cancer Epidemiology Biomarkers & Prevention. 10(1): 17-23.

Albanes D, Malila N, Taylor PR et al. (2000) Effects of supplemental alpha-
tocopherol and beta-carotene on colorectal cancer: results from a controlled trial
(Finland). Cancer Causes & Control. 11(3): 197-205.

Wu K, Willett WC, Chan JM et al. (2002) A prospective study on supplemental
vitamin E intake and risk of colon cancer in women and men. Cancer
Epidemiology Biomarkers & Prevention. 11(11): 1298-1304.

Malila N, Virtamo J, Virtanen M et al. (2002) Dietary and serum alpha-tocopherol,

beta-carotene and retinol, and risk for colorectal cancer in male smokers. European
Journal of Clinical Nutrition. 56(7): 615-621.

47

163.

164.

165.

166.

167.

168.

169.

170.

171.

172.

173.

Chung H, Wu DY, Han SN et al. (2003) Vitamin E supplementation does not alter
azoxymethane-induced colonic aberrant crypt foci formation in young or old mice.
Journal of Nutrition. 133(2): 528-532.

Wang WQ and Higuchi CM. (1995) Induction of NAD(P)H:quinone reductase by
vitamins A, E and C in 0010205 colon cancer cells. Cancer Letters. 98(1): 63-69.

Guyonnet D, Siess MH, Le Bon AM and Suschetet M. (1999) Modulation of phase
II enzymes by organosulfur compounds from allium vegetables in rat tissues.
Toxicology and Applied Pharmacology. 154(1): 50-58.

Wilkinson J and Clapper ML. (1997) Detoxication enzymes and chemoprevention.
Proceedings of the Society for Experimental Biology and Medicine. 216(2): 192-
200.

Munday R and Munday CM. (2001) Relative activities of organosulfur compounds
derived from onions and garlic in increasing tissue activities of quinone reductase
and glutathione transferase in rat tissues. Nutrition and Cancer-an International
Journal. 40(2): 205-210.

Begleiter A, Sivananthan K, Curphey TJ and Bird RP. (2003) Induction of
NAD(P)H quinone : oxidoreductasel inhibits carcinogen-induced aberrant crypt

foci in colons of Sprague-Dawley rats. Cancer Epidemiology Biomarkers &
Prevention. 12(6): 566-572.

St. Clair WH, Billings PC and Kennedy AR. ( 1990) The Effects of the Bowman-
Birk Protease Inhibitor on C-Myc Expression and Cell-Proliferation in the
Unirradiated and Irradiated Mouse Colon. Cancer Letters. 52(2): 145-152.

Chang JD, Li JH, Billings PC and Kennedy AR. (1990) Effects of Protease
Inhibitors on C-Myc Expression in Normal and Transformed C3h-10t1/2 Cell-
Lines. Molecular Carcinogenesis. 3(4): 226-232.

Billings PC and Habres JM. (1992) A Growth-Regulated Protease Activity That Is
Inhibited by the Anticarcinogenic Bowman-Birk Protease Inhibitor. Proceedings of
the National Academy of Sciences of the United States of America. 89(7): 3120-
3124.

Mutoh H, Takahashi M, Fukuda K et al. (2000) Suppression by flavonoids of
cyclooxygenase-2 promoter-dependent transcriptional activity in colon cancer cells:

Structure-activity relationship. Japanese Journal of Cancer Research. 91(7): 686-
691.

Wenzel H, Kuntz S, Brendel MD and Daniel H. (2000) Dietary flavone is a potent

apoptosis inducer in human colon carcinoma cells. Cancer Research. 60(14): 3823-
3831.

48

174.

175.

176.

177.

178.

179.

180.

181.

182.

183.

184.

185.

Surh YJ, Chun KS, Cha HH et al. (2001) Molecular mechanisms underlying
Chemopreventive activities of anti-inflammatory phytochemicals: down-regulation
of COX-2 and iNOS through suppression of NF-kappa B activation. Mutation
Research-Fundamental and Molecular Mechanisms of Mutagenesis. 480(243-268.

Banerjee T, Van der Vliet A and Ziboh VA. (2002) Downregulation of COX-2 and
iNOS by amentoflavone and quercetin in A549 human lung adenocarcinoma cell
line. Prostaglandins Leukotrienes and Essential Fatty Acids. 66(5-6): 485-492.

Cheong E, Ivory K, Doleman J et al. (2004) Synthetic and naturally occurring
COX-2 inhibitors suppress proliferation in a human oesophageal adenocarcinoma

cell line (OE33) by inducing apoptosis and cell cycle arrest. Carcinogenesis.
25(10): 1945-1952.

Pamaud G, Li PF, Cassar G et al. (2004) Mechanism of sulforaphane-induced cell
cycle arrest and apoptosis in human colon cancer cells. Nutrition and Cancer-an
International Journal. 48(2): 198-206.

Jackson SJT and Singletary KW. (2004) Sulforaphane: a naturally occurring
mammary carcinoma mitotic inhibitor, which disrupts tubulin polymerization.
Carcinogenesis. 25(2): 219-227.

Bingharn S. (1990) Mechanisms and experimental and epidemiological evidence
relating dietary fibre (non-starch polysaccharides) and starch to protection against
large bowel cancer. Proc. Nutr. Soc. 49(2): 152-157.

Reddy BS. (1992) Effect of dietary fiber on colonic bacterial enzymes and bile
acids in relation to colon cancer. Gastroenterology. 102: 1475-82.

Cummings JH. (1981) Short chain fatty acids in the human colon. Gut. 22(9): 763-
79.

Cummings JH and Macfarlane GT. (1991) The control and consequences of
bacterial fermentation in the human colon. J. Appl. Bacter. 70: 443-59.

Heerdt BG, Houston MA and Augenlicht LH. (1997) Short-chain fatty acid-
initiated cell cycle arrest and apoptosis of colonic epithelial cells is linked to
mitochondrial function. Cell Growth & Diflerentiation. 8: 523-32.

Hague A, Manning AM, Huschtscha LI et al. (1993) Sodium butyrate induces
apoptosis in human colonic tumor cell lines in a p-53 independent pathway:

implications for the possible role of dietary fiber in the prevention of large bowel
cancer. Int. J. Cancer. 55: 498-505.

Whitehead RH, Young GP and Bhatal PS. (1986) Effects of Short-Chain Fatty
Acids on a new human colon carcinoma cell line (LIMlZlS). Gut. 27:1457-63.

49

186.

187.

188.

189.

190.

191.

192.

193.

194.

195.

196.

197.

Thorup 1, Meyer O and Kristiansen E. (1995) Effects of potato starch, cornstarch,
and sucrose on aberrant crypt foci in rats exposed to azoxymethane.
AnticancerResearch. 1 5: 2101-2106.

Alabaster O, Tang ZC, Frost A and Shivapurkar N. (1995) Effect of Beta-Carotene
and Wheat Bran Fiber on Colonic Aberrant Crypt and Tumor-Formation in Rats
Exposed to Azoxymethane and High Dietary-F at. Carcinogenesis. 16(1): 127-132.

Madar Z, Weiss 0, Timar B et al. (1996) The effects of high-fiber diets on
chemically induced colon cancer in rats. Cancer Journal. 9(4): 207-211.

Sakamoto J, Nakaji S, Sugawara K et al. (1996) Comparison of resistant starch
with cellulose diet on 1,2-dimethylhydrazine-induced colonic carcinogenesis in
rats. Gastroenterology. 110(1): 116-120.

Young GP, McIntyre A, Albert V et al. (1996) Wheat bran suppresses potato
starch-potentiated colorectal tumorigenesis at the aberrant crypt stage in a rat
model. Gastroenterology. 110(2): 508-514.

Zoran DL, Turner ND and Taddeo SS. (1997) Wheat bran diet reduces tumor
incidence in a rat model of colon cancer independent of effects on distal luminal
butyrate concentrations. Journal of Nutrition. 127: 2217-25.

Cassand, Pierrette, Maziere S et al. (1997) Effects of resistant starch and Vitamin
A-supplemented diets on the promotion of precursor lesions of colon cancer in rats.
Nutrition and Cancer. 27(1): 53-59.

Pierre F, Perrin P, Champ M et al. (1997) Short-chain fructo-oligosaccharides
reduce the occurrence of colon tumors and gut-associated lymphoid tissue in Min
mice. Cancer Res.57: 225-28.

Alabaster O, Tang Z and Shivapurkar N. (1997) Inhibition by wheat bran cereals of
the development of aberrant crypt foci and colon tumours. Food and Chemical
Toxicology. 35(5): 517-522.

Madar Z, Gurevich P, Ben-Hur H et al. (1998) Effects of dietary fiber on the rat
intestinal mucosa exposed to low doses of a carcinogen. Anticancer Research.
18(5A): 3521-3526.

Rao CV, Chou D, Simi B et al. (1998) Prevention of colonic aberrant crypt foci and
modulation of large bowel microbial activity by dietary coffee fiber, inulin and
pectin. Carcinogenesis. 19(10): 1815-1819.

Verghese M, Rao DR, Chawan CB and Shackelford L. (2002) Dietary inulin

suppresses azoxymethane-induced preneoplastic aberrant crypt foci in mature
Fisher 344 rats. Journal of Nutrition. 132(9): 2804-2808.

50

198.

199.

200.

201.

202.

203.

204.

205.

206.

207.

208.

Compher CW, Frankel WL, Tazelaar J et al. (1999) Wheat bran decreases aberrant
crypt foci, preserves normal proliferation, and increases luminal butyrate levels in
experimental colon cancer. JP.E.N. 23: 269-78.

Lindner MA. (1991) A Fish Oil Diet Inhibits Colon Cancer in Mice. Nutrition and
Cancer-an International Journal. 15(1): 1-11.

Chang WCL, Chapkin RS and Lupton JR. (1997) Predictive value of proliferation,
differentiation and apoptosis as intermediate markers for colon tumorigenesis.
Carcinogenesis. 18(4): 721 -730.

Singh J, Hamid R and Reddy BS. (1998) Dietary fish oil inhibits the expression of
famesyl protein transferase and colon tumor development in rodents.
Carcinogenesis. 19(6): 985-989.

Chang WCL, Chapkin RS and Lupton JR. (1998) Fish oil blocks azoxymethane-
induced rat colon tumorigenesis by increasing cell differentiation and apoptosis
rather than decreasing cell proliferation. Journal of Nutrition. 128(3): 491-497.

Coleman LJ, Landstrom EK, Royle PJ et al. (2002) A diet containing alpha-
cellulose and fish oil reduces aberrant crypt foci formation and modulates other

possible markers for colon cancer risk in azoxymethane-treated rats. Journal of
Nutrition. 132(8): 2312-2318.

Covert KL, Newton AH, Turner ND et al. (2003) The combination of dietary fish
oil and butyrate decreases high-multiplicity aberrant crypt foci in experimentally
induced colon cancer. Faseb Journal. 17(5): A1154-A1154.

Singh J, Hamid R and Reddy BS. (1997) Dietary fat and colon cancer: Modulating
effect of types and amount of dietary fat on ras-p21 function during promotion and
progression stages of colon cancer. Cancer Research. 57(2): 253-258.

Davidson LA, Lupton JR, Jiang YH and Chapkin RS. (1999) Carcinogen and
dietary lipid regulate ras expression and localization in rat colon without affecting
famesylation kinetics. Carcinogenesis. 20(5): 785-791.

Collett ED, Davidson LA, Fan YY et al. (2001) n-6 and n-3 polyunsaturated fatty
acids differentially modulate oncogenic Ras activation in colonocytes. American
Journal of Physiology-Cell Physiology. 280(5): C1066-C1075.

Murray NR, Weems C, Chen L et al. (2002) Protein kinase C beta 11 and TGF beta

RII in omega-3 fatty acid-mediated inhibition of colon carcinogenesis. Journal of
Cell Biology. 157(6): 915-920.

51

209.

210.

211.

212.

213.

214.

215.

216.

217.

218.

Bartram HP, Gostner A, Scheppach W et al. (1993) Effects of Fish-Oil on Rectal
Cell-Proliferation, Mucosal Fatty-Acids, and Prostaglandin-E2 Release in Healthy-
Subjects. Gastroenterology. 105(5): 1317-1322.

Rao CV, Simi B, Wynn TT et al. (1996) Modulating effect of amount and types of
dietary fat on colonic mucosal phospholipase A(2), phosphatidylinositol-specific
phospholipase C activities, and cyclooxygenase metabolite formation during

different stages of colon tumor promotion in male F344 rats. Cancer Research.
56(3): 532-537.

Sandhu MS, Dunger DB and Giovannucci EL. (2002) Insulin, insulin-like grth
factor-I (IGF-I), IGF binding proteins, their biologic interactions, and colorectal
cancer. Journal of the National Cancer Institute. 94(13): 972-980.

Palmqvist R, Hallmans G, Rinaldi S et al. (2002) Plasma insulin-like growth factor
1, insulin-like grth factor binding protein 3, and risk of colorectal cancer: a
prospective study in northern Sweden. Gut. 50(5): 642-646.

Renehan AG, O'Connell J, O'Halloran D et al. (2003) Acromegaly and colorectal
cancer: A comprehensive review of epidemiology, biological mechanisms, and
clinical implications. Hormone and Metabolic Research. 35(11-12): 712-725.

Macdonald RS, Thornton WH and Bean TL. (1993) Insulin and Igf-l Receptors in
a Human Intestinal Adenocarcinoma Cell-Line (Caco-2) - Regulation of Na+

Glucose-Transport across the Brush-Border. Journal of Receptor Research. 13(7):
1093-1 1 l3.

Guo YS, Narayan S, Yallampalli C and Singh P. (1992) Characterization of
Insulin-Like Growth Factor-1 Receptors in Human Colon Cancer.
Gastroenterology. 102(4): 1 101 -1 108.

Ouban A, Muraca P, Yeatman T and Coppola D. (2003) Expression and

distribution of insulin-like growth factor-1 receptor in human carcinomas. Human
Pathology. 34(8): 803-808.

Koenuma M, Yamori T and Tsuruo T. (1989) Insulin and Insulin-Like Growth
Factor-I Stimulate Proliferation of Metastatic Variants of Colon Carcinoma-26.
Japanese Journal of Cancer Research. 80(1): 51-58.

Bjork J, Nilsson J, Hultcrantz R and Johansson C. (1993) Growth-Regulatory
Effects of Sensory Neuropeptides, Epidermal Growth-Factor, Insulin, and
Somatostatin on the Nontransformed Intestinal Epithelial-Cell Line Iec-6 and the
Colon-Cancer Cell-Line Ht-29. Scandinavian Journal of Gastroenterology. 28(10):
879-884.

52

219.

220.

221.

222.

223.

224.

225.

226.

227.

228.

229.

230.

Warren RS, Yuan H, Matli MR, Ferrara N and Donner DB. (1996) Induction of
vascular endothelial grth factor by insulin-like grth factor 1 in colorectal
carcinoma. Journal of Biological Chemistry. 271(46): 29483-29488.

Sekhararn M, Zhao HY, Sun M et al. (2003) Insulin-like grth factor 1 receptor
enhances invasion and induces resistance to apoptosis of colon cancer cells through
the Akt/Bcl-x(L) pathway. Cancer Research. 63(22): 7708-7716.

Corpet DE, Peiffer G and Tache S. (1998) Glycemic index, nutrient density, and
promotion of aberrant crypt foci in rat colon. Nutrition and Cancer-an
International Journal. 32(1): 29-36.

Tran TT, Medline A and Bruce WR. (1996) Insulin promotion of colon tumors in
rats. Cancer Epidemiology Biomarkers & Prevention. 5(12): 1013-1015.

Koohestani N, Chia MC, Pham NA et al. (1998) Aberrant crypt focus promotion
and glucose intolerance: correlation in the rat across diets differing in fat, n-3 fatty
acids and energy. Carcinogenesis. 19(9): 1679-1684.

Geil PB and Anderson JW. (1994) Nutrition and Health Implications of Dry Beans
- a Review. Journal of the American College of Nutrition. 13(6): 549-558.

Messina MJ. (1999) Legumes and soybeans: overview of their nutritional profiles
and health effects. American Journal of Clinical Nutrition. 70(3): 439S-450S.

Anderson JW, Smith BM and Washnock CS. (1999) Cardiovascular and renal
benefits of dry bean and soybean intake. American Journal of Clinical Nutrition.
70(3): 464S-474S.

Setchell KDR and Radd S. (2000) Soy and other legumes: 'Bean' around a long
time but are they the 'superfoods' of the millennium and what are the safety issues

for their constituent phytoestrogens? Asia Pacific Journal of Clinical Nutrition.
9:Sl3-S22.

Messina M and Messina V. (1991) Increasing Use of Soyfoods and Their Potential

Role in Cancer Prevention. Journal of the American Dietetic Association. 91(7):
836-840.

Endres JG. (2001) Soy protein products: Characteristics, Nutritional Aspects, and
Utilization, (Endres JG, ed.), AOCS Press, Champaign, IL, pp. 4-40.

US Department of Agriculture, Agricultural Research Service. (1999) USDA

Nutrient database for standard reference, release 13. Nutrient data laboratory home
page, www.nal.usdagov/fnic/foodcomp.

53

231.

232.

233.

234.

235.

236.

237.

238.

239.

240.

241.

242.

Kushi LH, Meyer KA and Jacobs DR. (1999) Cereals, legumes, and chronic
disease risk reduction: evidence from epidemiologic studies. American Journal of
Clinical Nutrition. 70(3): 4518-4588.

Ludwig DDS. (2002) The glycemic index - Physiological mechanisms relating to
obesity, diabetes, and cardiovascular disease. Jama-Journal of the American
Medical Association. 287(18): 2414-2423.

Simpson HCR, Lousley S, Geekie M et al. (1981) A High-Carbohydrate
Leguminous Fiber Diet Improves All Aspects of Diabetic Control. Lancet. 1(8210):
1-4.

Jenkins DJA, Wolever TMS, Jenkins AL et a1. (1983) The Glycemic Index of
Foods Tested in Diabetic-Patients - a New Basis for Carbohydrate Exchange
F avoring the Use of Legumes. Diabetologia. 24 (4): 257-264.

Dilawari JB, Kumar VKA, Khurana S et al. (1987) Effect of Legumes on Blood-
Sugar in Diabetes-Mellitus. Indian Journal of Medical Research. 85: 184-187.

Viswanathan M, Ramachandran A, Indira P et al. (1989) Responses to Legumes in
Niddrn Subjects - Lower Plasma-Glucose and Higher Insulin Levels. Nutrition
Reports International. 40(4): 803-812.

Jenkins DJA, Wong GS, Patten R et al. (1983) Leguminous Seeds in the Dietary-
Management of Hyperlipidemia. American Journal of Clinical Nutrition. 38(4):
567-573.

Hu FB, Rimm EB, Stampfer MJ et al. (2000) Prospective study of major dietary

patterns and risk of coronary heart disease in men. American Journal of Clinical
Nutrition. 72: 912-921.

Bazzano LA, He J, Ogden LG et al. (2001) Legume consumption and risk of
coronary heart disease in US men and women. Archives of Internal Medicine. 161:
2573-2578.

Fung TT, Willett WC, Stampfer MJ et al. (2001) Dietary patterns and the risk of
coronary heart disease in women. Archives of Internal Medicine. 161: 1857-1862.

Bazzano LA, He J, Ogden LG et al. (2001) Legume consumption and risk of
coronary heart disease in US men and women. Archives of Internal Medicine.
161(21): 2573-2578.

Leathwood P and Pollet P. (1988) Effects of slow release carbohydrates in the form
of bean flakes on the evolution of hunger and satiety in man. Appetite. 10: 1-11.

54

243.

244.

245.

246.

247.

248.

249.

250.

251.

252.

253.

254.

255.

Ludwig DS, Majzoub JA, A1 Zahrani A et al. (1999) High glycemic index foods,
overeating, and obesity. Pediatrics. 103(3):

Ludwig DS, Spieth LE, Lenders CM et al. (2000) A low glycemic index diet in the
treatment of obesity. Pediatric Research. 47(4): 134A-134A.

Steinmetz KA and Potter JD. (1993) Food-group consumption and colon cancer in
the Adelaide Case-Control Study. I. Vegetables and fruit.
IntJCancer. 53(5): 711-9.

Benito E, Stiggelbout A, Bosch FX et al. (1991) Nutritional factors in colorectal
cancer risk: a case-control study in Majorca. Int J Cancer. 49(2): 161-7.

Messina M and Bennink MR. (1998) Soyfoods, isoflavones and risk of colonic
cancer: a review of the in vitro and in vivo data.
Baillieres Clin Endocrinol Metab. (4): 707-28.

Spector D, Anthony M, Alexander D and Arab L. (2003) Soy consumption and
colorectal cancer. Nutr Cancer. 47(1): 1-12.

Nishi M, Yoshida K, Hirata K, and Miyake H. (1997) Eating habits and colorectal
cancer. Oncol Reports. 4: 995-998.

Nagata C. (2000) Ecological study of the association between soy product intake
and mortality from cancer and heart disease in Japan. Int Epidemiol Assoc. 29:
832—836.

McKeown-Eyssen GE and Bright-See E. (1984) Dietary factors in colon cancer:
international relationships. Nutr Cancer. 6: 160—170.

Haenszel W, Berg JW, Segi M et al. (1973) Large bowel cancer in Hawaiian
Japanese. JNCI. 51: 1765-1779.

Tajima K and Tominaga S. (1985) Dietary habits and gastro-intestinal cancers: a
comparative case-control study of stomach and large intestinal cancers in Nagoya,
Japan. Jpn J Cancer Res. 76: 705—716.

Hirayama T. (1990) Contribution of a long-term prospective cohort study to the
issue of Nutrition and cancer with special reference to the role of alcohol drinking.
Prog Clin Biol Res. 346: 179—187.

Hu J, Liu Y, Yu Y et al. (1991) Diet and cancer of the colon and rectum: a case-
control study in China. Int J Epidemiol. 20: 362—367.

55

256.

257.

258.

259.

260.

261.

262.

263.

264.

265.

266.

267.

Hoshiyama Y, Sekine T and Sasaba T. (1993) A case-control study of colorectal
cancer and its relation to diet, cigarettes, and alcohol consumption in Saitama
Prefecture, Japan. T ohoku J Exp Med. 171: 153—165.

Kono S, Irnanishi K, Shinchi K and Yanai F. (1993) Relationship of diet to small
and large adenomas of the sigmoid colon. Jpn J Cancer Res. 84: 13—19.

Inoue M, Tajima K, Hirose K et al. (1995) Subsite-specific risk factors for
colorectal cancer: a hospital-based case-control study in Japan. Cancer Cause
Control. 6: 14—22.

Witte J S, Longnecker MP, Bird CL et al. (1996) Relation of vegetable, fruit, and
grain consumption to colorectal adenomatous polyps. Am J Epidemiol. 144: 1015—
1025.

Le Marchand L, Hankin JH, Wilkens LR et al. (1997) Dietary fiber and colorectal
cancer risk. Epidemiology. 8: 658—665.

Nishi M, Yoshida K, Hirata K, and Miyake H. (1997) Eating habits and colorectal
cancer. Oncol Rep. 4: 995—998.

Bennink MR. (2001) Dietary soy reduces colon carcinogenesis in human and rats -
Soy and colon cancer. Nutrition and Cancer Prevention. 492: 11-17.

Thiagarajan DG, Bennink MR, Bourquin LD and Kavas FA. ( 1998) Prevention of
precancerous colonic lesions in rats by soy flakes, soy flour, genistein, and calcium.
American Journal of Clinical Nutrition. 68(6): 1394S-1399S.

McIntosh GH, Regester GO, Leleu RK et al. (1995) Dairy Proteins Protect against
Dimethylhydrazine-Induced Intestinal Cancers in Rats. Journal of Nutrition.
125(4): 809-816.

Rondini EA and Bennink MR. (2002) Defatted soy flour, but not soy concentrate,

reduces azoxymethane-induced colon carcinogenesis. Journal of Nutrition. 132(3):
589S-589S.

Helms JR and Gallagher DD. (1995) The effect of dietary soy protein isolate and
genistein on the development of preneoplastic lesions (aberrant crypts) in rats.
Journal of Nutrition. 125: 8028.

Gallagher DD, Gallagher CM, An ZL and Hoffman RM. (1998) Soy protein

isolates and genistein: effects on initiation, promotion, and progression of colon
cancer. American Journal of Clinical Nutrition. 68(6): 1524S-1524S.

56

268.

269.

270.

271.

272.

273.

274.

275.

276.

277.

278.

279.

Gee JM, Notebom H, Polley ACJ and Johnson IT. (2000) Increased induction of
aberrant crypt foci by 1,2- dimethylhydrazine in rats fed diets containing purified
genistein or genistein-rich soya protein. Carcinogenesis. 21(12): 2255-2259.

Hakkak R, Korourian S, Ronis MJJ et al. (2001) Soy protein isolate consumption
protects against azoxymethane- induced colon tumors in male rats. Cancer Letters.
166(1): 27-32.

Weed HG, McGandy RB and Kennedy AR. (1985) Protection against
Dimethylhydrazine-Induced Adenomatous Tumors of the Mouse Colon by the
Dietary Addition of an Extract of Soybeans Containing the Bowman-Birk Protease
Inhibitor. Carcinogenesis. 6(8): 1239-1241.

Billings PC, Newbeme PM and Kennedy AR. (1990) Protease Inhibitor
Suppression of Colon and Anal Gland Carcinogenesis Induced by
Dimethylhydrazine. Carcinogenesis. 11(7): 1083-1086.

Kennedy AR, Billings PC, Wan XS and Newbeme PM. (2002) Effects of Bowman-
Birk inhibitor on rat colon carcinogenesis. Nutrition and Cancer-an International
Journal. 43(2): 174-186.

Shamsuddin AM, Elsayed AM and Ullah A. (1988) Suppression of Large Intestinal
Cancer in F344 Rats by Inositol Hexaphosphate. Carcinogenesis. 9(4): 577-580.

Shamsuddin AM and Ullah A. (1989) Inositol Hexaphosphate Inhibits Large
Intestinal Cancer in F344 Rats 5 Months after Induction by Azoxymethane.
Carcinogenesis. 10(3): 625-626.

Ullah A and Shamsuddin AM. (1990) Dose-Dependent Inhibition of Large
Intestinal Cancer by Inositol Hexaphosphate in F344 Rats. Carcinogenesis. 11(12):
2219-2222.

Graf E and Eaton JW. (1993) Suppression of Colonic-Cancer by Dietary Phytic
Acid. Nutrition and Cancer-an International Journal. 19(1): 11-19.

Rao CV, Wang CX, Simi B et al. (1997) Enhancement of experimental colon
cancer by genistein. Cancer Research. 57(17): 3717-3722.

Sorensen IK, Kristiansen E, Mortensen A et al. (1998) The effect of soy
isoflavones on the development of intestinal neoplasia in Apc(Min) mouse. Cancer
Letters. 130(1-2): 217-225.

Guo JY, Li XS, Browning JD et al. (2004) Dietary soy isoflavones and estrone
protect ovariectomized ER alpha KO and wild-type mice from carcinogen-induced
colon cancer. Journal of Nutrition. 134(1): 179-182.

57

280.

281.

282.

283.

284.

285.

286.

287.

288.

289.

290.

Koratkar R and Rao AV. (1997) Effect of soya bean saponins on azoxymethane-
induced preneoplastic lesions in the colon of mice. Nutrition and Cancer-an
International Journal. 27(2): 206-209.

Raicht RF, Cohen BI, Fazzini EP et al. (1980) Protective effect of plant sterols
against chemically induced colon tumors in rats. Cancer Research. 40(2): 403-5.

Witschi H and Kennedy AR. (1989) Modulation of Lung-Tumor Development in
Mice with the Soybean-Derived Bowman-Birk Protease Inhibitor. Carcinogenesis.
10(12): 2275-2277.

Vucenik I, Sakamoto K, Bansal M and Shamsuddin AM. (1993) Inhibition of Rat
Mammary Carcinogenesis by Inositol Hexaphosphate (Phytic Acid) — a Pilot-Study.
Cancer Letters. 75(2): 95-102.

Vucenik I, Yang GY and Shamsuddin AM. (1995) Inositol Hexaphosphate and
Inositol Inhibit Dmba-Induced Rat Mammary-Cancer. Carcinogenesis. 16(5):
1055-1058.

Vucenik I, Yang G and Shamsuddin AM. (1997) Comparison of pure inositol
hexaphosphate and high-bran diet in the prevention of DMBA-induced rat
mammary carcinogenesis. Nutrition and Cancer-an International Journal. 28(1): 7-
13.

Estensen RD and Wattenberg LW. (1993) Studies of Chemopreventive Effects of
Myoinositol on Benzo[a]Pyrene-Induced Neoplasia of the Lung and Forestomach
of Female a/J Mice. Carcinogenesis. 14(9): 1975-1977.

Gupta KP, Singh J and Bharathi R. (2003) Suppression of DMBA-induced mouse
skin tumor development by inositol hexaphosphate and its mode of action.
Nutrition and Cancer-an International Journal. 46(1): 66—72.

Vucenik 1, Zhang ZS and Shamsuddin AM. (1998) IP6 in treatment of liver cancer
11. Intra-tumoral injection of 1P6 regresses pre-existing human liver cancer
xenotransplanted in nude mice. Anticancer Research. 18(6A): 4091-4096.

Vucenik I, Tomazic VJ, Fabian D and Shamsuddin AM. (1992) Antitumor-Activity
of Phytic Acid (Inositol Hexaphosphate) in Murine Transplanted and Metastatic
Fibrosarcoma, a Pilot-Study. Cancer Letters. 65(1): 9-13.

Billings PC, Stclair WH, Maki PA and Kennedy AR. (1992) Distribution of the

Bowman Birk Protease Inhibitor in Mice Following Oral-Administration. Cancer
Letters. 62(3): 191-197.

58

291.

292.

293.

294.

295.

296.

297.

298.

299.

300.

301.

Billings PC, Brandon DL and Habres JM. (1991) Intemalization of the Bowman-
Birk Protease Inhibitor by Intestinal Epithelial-Cells. European Journal of Cancer.
27(7): 903-908.

Messadi DV, Billings P, Shklar G and Kennedy AR. (1986) Inhibition of Oral
Carcinogenesis by a Protease Inhibitor. Journal of the National Cancer Institute.
76(3): 447-452.

Stclair WH, Billings PC and Kennedy AR. (1990) The Effects of the Bowman-Birk
Protease Inhibitor on C-Myc Expression and Cell-Proliferation in the Unirradiated
and Irradiated Mouse Colon. Cancer Letters. 52(2): 145-152.

Vucenik I and Shamsuddin AM. (2003) Cancer inhibition by inositol
hexaphosphate (1P6) and inositol: From laboratory to clinic. Journal of Nutrition.
133(11): 37788-3784S.

Sandra F, Matsuda M, Yoshida H and Hirata M. (2002) Inositol hexakisphosphate
blocks tumor cell growth by activating apoptotic machinery as well as by inhibiting

the Akt/NF kappa B-mediated cell survival pathway. Carcinogenesis. 23(12):
2031-2041.

El-Sherbiny YM, Cox MC, Ismail ZA et al. (2001) G(O)/G(1) arrest and S phase
inhibition of human cancer cell lines by inositol hexaphosphate (1P6). Anticancer
Research. 21(4A): 2393-2403.

Singh RP, Agarwal C and Agarwal R. (2003) Inositol hexaphosphate inhibits
growth, and induces G1 arrest and apoptotic death of prostate carcinoma DU145
cells: modulation of CDKI-CDK-cyclin and pr-related protein-E2F complexes.
Carcinogenesis. 24(3): 555-563.

Jariwalla RJ, Sabin R, Lawson S et al. (1999) Dietary intrinsic phytate protects
colon from lipid peroxidation in pigs with a moderately high dietary iron intake.
Proceedings of the Society for Experimental Biology and Medicine. 221(1): 80-86.

Nelson RL. (1992) Dietary Iron and Colorectal-Cancer Risk. Free Radical Biology
and Medicine. 12(2): 161-168.

Pagliacci MC, Smacchia M, Migliorati G et al. (1994) Growth-Inhibitory Effects of
the Natural Phyto-Estrogen Genistein in Mcf-7 Human Breast-Cancer Cells.
European Journal of Cancer. 30A(11): 1675-1682.

Peterson G and Barnes S. (1996) Genistein inhibits both estrogen and growth

factor-stimulated proliferation of human breast cancer cells. Cell Growth &
Differentiation. 7(10): 1345-1351.

59

302.

303.

304.

305.

306.

307.

308.

309.

310.

311.

312.

313.

Choi YH, Zhang LJ, Lee WH and Park KY. ( 1998) Genistein-induced G2/M arrest
is associated with the inhibition of cyclin B1 and the induction of p21 in human
breast carcinoma cells. International Journal of Oncology. 13(2): 391-396.

Zhou JR, Mukherjee P, Gugger ET et al. (1998) Inhibition of murine bladder
tumorigenesis by soy isoflavones via alterations in the cell cycle, apoptosis, and
angiogenesis. Cancer Research. 58(22): 5231-5238.

Li YW, Upadhyay S, Bhuiyan M and Sarkar FH. ( 1999) Induction of apoptosis in
breast cancer cells MDA-MB-231 by genistein. Oncogene. 18(20): 3166-3172.

Salti GI, Grewal S, Mehta R et al. (2000) Genistein induces apoptosis and
topoisomerase II-mediated DNA breakage in colon cancer cells. European Journal
of Cancer. 36(6): 796-802.

Nguyen DT, Garcia J and Cadenas E. (2001) Genistein causes cell cycle arrest and
apoptosis via inhibition of p53-independent mechanisms in T47D breast cancer
cells. Free Radical Biology and Medicine. 31(S105-8105.

Yu ZL, Li WJ and Liu FY. (2004) Inhibition of proliferation and induction of
apoptosis by genistein in colon cancer HT-29 cells. Cancer Letters. 215(2): 159-
166.

Yu ZL, Tang YN, Hu DS and L1 J. (2005) Inhibitory effect of genistein on mouse
colon cancer MC-26 cells involved TGF-beta l/Smad pathway. Biochemical and
Biophysical Research Communications. 333(3): 827-832.

Kerry N and Abbey M. (1998) The isoflavone genistein inhibits copper and peroxyl
radical mediated low density lipoprotein oxidation in vitro. Atherosclerosis. 140(2):
341-347.

Boadi WY, Iyere PA and Adunyah SE. (2005) In vitro exposure to quercetin and
genistein alters lipid peroxides and prevents the loss of glutathione in human
progenitor mononuclear (U937) cells. Journal of Applied Toxicology. 25(1): 82-88.

Fotsis T, Pepper M, Adlercreutz H et al. (1993) Genistein, a dietary-derived
inhibitor of in vitro angiogenesis. Proc Natl Acad Sci U S A. 90: 2690-2694

Fotsis T, Pepper MS, Aktas E et al. (1997) Flavonoids, dietary-derived inhibitors of
cell proliferation and in vitro angiogenesis. Cancer Research. 57: 2916-2921.

Wietrzyk J, Boratynski J, Grynkiewicz G et al. (2001) Antiangiogenic and

antitumour effects in vivo of genistein applied alone or combined with
cyclophosphamide. Anticancer Research. 21(6A): 3893-3896.

60

314.

315.

316.

317.

Buchler P, Reber HA, Buchler MW et al. (2004) Antiangiogenic activity of
genistein in pancreatic carcinoma cells is mediated by the inhibition of hypoxia-

inducible factor-1 and the down-regulation of VEGF gene expression. Cancer.
100(1): 201-210.

Akiyama T, Ishida J, Nakagawa S et al. (1987) Genistein, a specific inhibitor of
tyrosine-specific protein kinases. J Biol Chem. 262: 5592-5595.

Davidson LA, Nguyen DV, Hokanson RM et al. (2004) Chemopreventive n-3
polyunsaturated fatty acids reprogram genetic signatures during colon cancer
initiation and progression in the rat. Cancer Res. 64(18): 6797-804.

Xiao R, Badger TM, Simmen FA. (2004) Dietary exposure to soy or whey proteins

alters colonic global gene expression profiles during rat colon tumorigenesis. Mol
Cancer. 4(1): 1-17.

61

CHAPTER III.
PROFILE OF GLOBAL GENE PATTERNS ALTERED IN

AZOXYMETHANE (AOM-INDUCED COLON TUMORS COMPARED TO
NORMAL-APPEARING MUCOSA

62

.L:._
I

5 ABSTRACT.

The azoxymethane (AOM)-treated rodent model has been utilized extensively for
studying the etiology and molecular pathogenesis of colon cancer. This study was
conducted to gain firrther insight into cellular and environmental differences in gene
changes underlying AOM-induced colon cancer progression using microarrays. Male
F344 rats were obtained at 3 weeks of age and fed a modified AIN-93G diet. At 4 and 5
weeks of age, animals were administered subcutaneous injections of AOM prepared in
saline (15 mg/kg) to induce colon tumors. Animals were sacrificed at 36 weeks of age
and microarrays were performed on mRNA isolated from tumorous tissue and
surrounding non-neoplastic mucosa. Of the 8799 genes or ESTs present on RGU34A rat
genome chips, we identified 471 transcripts (306 over-expressed and 165 under-
expressed) significantly altered in tumors compared to normal colon tissue. A majority
of genes (15%) induced during cancer progression were related to inflammatory and
immune responses with evidence of tissue damage, remodeling, angiogenesis as well as a
lower production of cytokines involved in anti-tumor immunity. A lower expression of
ion transporters and metabolic enzymes highlights cellular disturbances in ion and water

absorption and oxidative mitochondrial metabolism during cancer progression.

63

2, INTRODUCTION.

The development of CRC is a multi-stage process characterized by the
accumulation of critical genetic and epigenetic events (1-2). For a majority of CRC,
these changes culminate in the transformation of normal epithelium to adenomatous
polyps and eventually malignant carcinomas (1-2). Critical mutations or gene deletions
involved in the adenoma-carcinoma sequence in humans have been identified (1-2) and
downstream signaling events are beginning to be explored in more detail (3-7).
Additionally the association of chronic inflammation to risk of colon cancer is well
documented (8-9), and the importance of communication and signaling between
colonocytes and the extracellular matrix in maintaining normal mucosal function is
becoming more recognized (4-7). However, despite being one of the earliest cancers by
which a genetic model was proposed, CRC remains the second leading cause of cancer
mortality in the US (10), indicating incomplete understanding of events involved in
neoplastic transformation and metastases.

A majority of sporadic CRC cases are thought to be attributable to environmental
exposures. Because the process of CRC can take up to 20 years in humans to fully
manifest, animals are frequently utilized to screen for and examine molecular
mechanisms whereby chemopreventative agents, including diet, can prevent or delay the
cancer process (12). Druckery and collegues (13-15) were first to demonstrate that
administration of the alkylating agent 1,2-dimethylhydrazine (DMH) or its active
metabolite, azoxymethane (AOM) specifically and reliably induces colon cancer in
rodents, and colon cancers induced in this model have been demonstrated to share similar

molecular and pathological features to those in humans (11, 16). Although APC

64

mutations are rare (17), mutational activation and nuclear translocation of B-catenin have
been reported (18). Additionally, mutations on codon 12 and 13 of the k-ras gene (19)
accompanied by up-regulation of cyclo-oxygenase 2 (COX-2) and inducible nitric oxide
synthase (iNOS) are other features common to human cancers and in the AOM model
(20-22).

This study was conducted to more thoroughly identify environmental and
molecular changes underlying AOM-induced colon cancer as well as potential
biomarkers involved in cancer progression. Male F344 rats, a highly susceptible strain,
were subject to a long-term carcinogenesis study, and at 31 weeks following AOM
administration, a time point when adenocarcinomas had developed, microarrays were
performed on tumor tissue and surrounding non-neoplastic mucosa. The profile of genes
differentially expressed and the biological relevance of some of the changes to human

colon cancer are discussed.

_g MATERIALS AND METHODS.
Experimental protocol and tissue collection.

The tissues utilized in this experiment were obtained from part of a larger colon
cancer study. Briefly, 3-week old male F 344 rats (Harlan Sprague-Dawley, Indianapolis,
IL) were fed a modified AIN-93G diet containing 11% dietary fiber, 16.7% fat, and
18.9% of protein as high nitrogen casein. After a one-week acclimatization period, rats
received subcutaneous injections of azoxymethane (AOM, 15 mg/kg) prepared in saline
once per week for two weeks. At 36 weeks of age rats were sacrificed and colons were
resected, opened longitudinally, and rinsed briefly in tap water to remove debris.

Polypoid lesions, when present, were excised from the distal half of the colon with a

65

razor blade. Epithelial cells were then collected by gently scraping the remaining
normal-appearing mucosa from the distal half of the colon with the edge of a glass slide.
All samples were snap frozen on dry ice immediately after collection and stored at —80 °C
until needed.

Throughout the experiment, animals had free access to diet and distilled water and
were assessed daily for health status and monthly for weight gain. The procedures used
in this study were in accordance with the regulatory guidelines of the Michigan State
University Committee on Animal Use and Care.

Microarray target preparation and hybridization.

For total RNA isolation, < 100 mg colonic mucosa or tumorous tissue was
homogenized using a Tekmar homogenizer in one mL of TRIzol reagent containing 2 1.1L
of RNase-free glycogen according to the manufacturers instructions (Gibco, Carlsbad,
CA). Following extraction, RNA was purified with RNeasy mini columns (Qiagen,
Valencia, CA), quantified using a UV spectrophotometer (A260/A280), and the quality
was assessed by agarose-formaldehyde gel electrophoresis. Prior to cDNA synthesis,
RNA was pooled from 2-4 animals/tissue type (tumors, normal mucosa) to control for
biological variation in gene expression.

Biotinylated cRNA was prepared from high quality RNA (10 pg) in accordance
with instructions supplied in the GeneChip Expression Manual (Affymetrix, Santa Clara,
CA). Approximately 15 ug cRNA was then fragmented at 94 °C for 35 minutes and
hybridized to RGU34A rat genome chips for 16 hours at 45 °C. Following hybridization,

arrays were washed and stained with a streptavidin-phycoerythrin conjugate on an

66

Affymetrix Fluidics station according to standard protocol. Processed arrays were
scanned at 570 nm using a Hewlett Packard GeneArray Scanner.
Quantitative reverse transcription-PCR (R T -PCR) analysis

The pooled RNA samples used for oligonucleotide microarrays were also used to
confirm gene changes with quantitative real time polymerase chain reaction (RT-PCR).
Gene specific primers for (B-Actin, group Ila secretory phospholipase A2 (sPLA2), NP
defensin 30L, lysozyme, and deleted in malignant brain tumors (DMBTl) were designed
with the Primer Express 2.0 program (TABLE 1; Applied Biosystems). Single-stranded
(ss) cDNA was synthesized from 2.5 pg of total RNA using T7-(dT)24 primers (Proligo,
Boulder, CO) and the Superscript II system (Invitrogen, Carlsbad, CA). Reverse
transcription was performed in a thermocycler following the Superscript first strand
synthesis protocol with the following modifications: temperature of reaction was cooled
to 20 °C for 5 minutes following annealing of primer and ss cDNA synthesis time was
increased to 60 minutes for 42 °C.

Quantitative determination of . gene expression was performed with the
ABIPrism7000 (Perkin Elmer Corp., Foster City, CA) using the Syber Green Universal
Master Mix (Applied Biosystems). The reaction mixture (25 uL total volume) contained
20 ng 55 cDNA, 12.5 uL SYBR Green RT-PCR Master mix, and 10.5 uL of diluted
primers (forward and reverse). Primer pairs were used at a final concentration of 1.2 uM.
The real-time cycle conditions were as follows: PCR initial activation step at 95 °C for 15
min and a total of 40 cycles for melting (95 °C, 15 s) and annealing/extension (60 °C, 1

min). All assays were performed in duplicates, using 2-3 samples per group.

67

 

Statistical analysis methods.

Data for microarrays and RT-PCR were analyzed using the General Linear Model
procedure of SAS (SAS Institute, Inc. Cary, NC, Version 7.0), and when appropriate,
individual comparisons were made using the least significant difference (LSD) method.
Prior to statistical analyses, microarrays were globally scaled to a target intensity of 500
in Affymetrix Microarray Suite, version 5.0 to control within-chip variations and then
imported into GeneSpring (Silicon Genetics, Inc., Redwood City, CA, Version 6.0) for
normalization and filtering. All chips were normalized to the median intensity of
invariant genes, defined as genes displaying less than a 30% coefficient of variation (CV)
across tissue types (mucosa, tumors). A series of filtering steps were then performed to
limit analysis of genes to those reliably measured and likely of physiological
significance. The first filtering step excluded genes not considered “Present” or
“Marginal” in at least 33% (2 out of 6) of the samples, reducing the gene list from 8799
to 4404 genes (50% of genes present on the chip). An additional filtering step limited the
genes further to those exhibiting greater than 2 fold- or less than 0.5 mean fold-change
difference between tissue types (875 genes). Normalized expression values were then
analyzed using the GLM procedure of SAS.

Differentially expressed transcripts (P < 0.05) were then broadly categorized
according to biological processes using gene ontologies retrieved from the Affymetrix
NetAffix Analysis Center (http://www.affymetrix.com/analysis/index.affx) and the Rat

Genome Database (RGD) gene annotation tool (Rat Genome Database Web Site, Medical

68

 

.2 HmEQV _ 8088 808 8080208 8 0000—00 080 3.0503 N< 0008—0800080 00.0005 ”0800030802 _

 

050.0800.000000000080500 80000000008000.5088 £020
5.000008005050050 5.0080000080800000 05%
00080500000005? 80000055000005 00 0000000 02
05300000008000.0000 00080008003300.8008 280003
00080008000000.0000 0<0<000<00<0<<<<00000<< 002-0

00-00 SEE 020:: 00-00 025.... 0.03.8.0 2.00

 

_ .mvméw— .50 000: 0.809 0008.:— ._ HAM—40h.

 

69

College of Wisconsin, Milwaukee, Wisconsin; World Wide Web: httgflrgdmcwedu/

 

gatool/). When present, duplicate entries representing the same transcript were averaged
following statistical analysis. All data are presented as relative mean fold-change

differences standardized to normal mucosal samples.

9; RESULTS.

We identified 472 transcripts (307 up-regulated, 165 down-regulated)
differentially expressed in AOM-induced colon cancers compared to normal-appearing
mucosa (P < 0.05). Genes with known identity/function (n=389) were broadly grouped
into one of fourteen categories and included:

1. Cell adhesion, cell communication

2. Cell cycle, cell grth & maintenance, apoptosis

3. Channel, transporter, carrier proteins

4. Cytoskeleton, microtubule processes, structural filaments
5. Electron transport, oxidoreductase, detoxification

6. Extracellular matrix, tissue regeneration, wound repair
7. Immune, defense, inflammatory response

8. Metabolism

9. Proteolysis and peptidolysis

10. RNA processing

11. Signal transduction

12. Transcription regulation, nucleic acid binding

13. Enzymes

70

14. Other

FIGURE 1 depicts the functional distribution of genes expressed as a percentage
of total known transcripts (n=389). Genes involved in immune, defense, and
inflammation (13%, n=50), other (15%, n=59), signal transduction (10%, n=40),
metabolism (7.2%, n=28), cell cycle, cell growth & maintenance, apoptosis (8.2%, n=32),
and channel, transporter, carrier proteins (8.5%, n=33) represented the majority of genes
changed between tissue types (mucosa vs. tumors).

A select list of differentially expressed transcripts is presented in TABLE 2, and a
full listing of the remaining transcripts can be found in the APPENDICES as
SUPPLEMENTARY TABLE 1. As shown in TABLE 2, genes involved in
inflammation, chemotaxis, immune responses, and extracellular matrix remodeling were
highly represented in colon cancers. Among specific genes repressed in tumor tissue
included metabolic enzymes (PEPCK, carbonic anhydrase) and ion transport/carrier
proteins as well as putative tumor suppressor genes (DMBTI, APC).

Quantitative R T -PCR.

RT-PCR was used to confirm gene changes for sPLA2, NP defensin 3a,
lysozyme, and deleted in malignant brain tumorsl (DMBTl) and results are presented in
FIGURE 2. There was general consistency in relative fold-changes with RT-PCR

compared to values obtained from microarrays.

71

 

Cell adhesion, cell communication ‘

Cell cycle, cell 8Y0Wth & maintenance, apoptosis V

Channel, transporter, carrier proteins ii,

Cytoskeleton, microtubule processes, structural

filaments
Electron transport, oxidoreductase, detoxification ~

Enzymes, other 5'2

Extracellular matrix, tissue regeneration, wound .
repair

 

 

 

 

 

‘ I % Increased E
l111'] % Decreased

., Fig—E W .._ ,

 

Immune, defense, irnflammatory response 3'

 

Metabolism . .

Proteolysis and peptidolysis {.1 P

RNA processing El

 

 

 

 

 

 

Signal Transduction

 

Transcription regulation, nucleic acid binding

 

 

 

 

Other

 

FIGURE 1. Biological classification of genes altered in AOM-induced colon

cancers. Values expressed as a percentage based on the number of genes changed in

5%

10%

 

 

 

15% 20%

Percentage of known genes

colon cancer for each category divided by the total number of known genes (n=3 89).

72

 

TABLE 2. Genes significantly altered in AOM-induced colon cancers relative to
normal colonic mucosa. l

 

 

Gene

my.“ Gene Title Tumor
1. Cell cycle, cell growth and maintenancg, apoptosis
Ptn pleiotrophin l 7
Igfbp3 insulin-like grth factor binding protein 3 13
--- lymphotoxin a 10
Sh3kbp1 sh3-domain kinase binding protein 1 10
Dab 2 disabled homolog 2, mitogen-responsive phosphoprotein 6.7

(drosophrla)
--- v-myc avian myelocytomatosis viral oncogene homolog 4.5
Ripk3 receptor-interacting serine-threonine kinase 3 4.4
Fbln5 fibulin 5 3.7
I d2 inhibitor of dna binding 2, dominant negative helix-loop-helix 3.6
protern

Naplll nucleosome assembly protein 1-like 1 3.2
Btg3 b-cell translocation gene 3 3.1
Tp53 tumor protein p53 3.0
Ngfrapl nerve grth factor receptor associated protein 1 2.9
Psmdl 26s proteasome, subunit p112 2.8
Cdk4 cyclin-dependent kinase 4 2.6
Ns nucleostemin 2.4
Etsl v-ets erythroblastosis virus e26 oncogene homolog 1 (avian) 2.3
Adprt ADP-ribosyltransferase 1 2.2
Pcna proliferating cell nuclear antigen 2.2
Fyn fyn proto-oncogene 2.2
--- cyclin e 2.2
Ccndl cyclin D1 2.1
Caspl caspase 1 0.5
Prkcd protein kinase c, delta 0.5
8th b-cell translocation gene 1 0.5

5pc adenomatosis polyposis coli 0.5

 

73

 

TABLE 2. Genes significantly altered in AOM-induced colon cancers relative to
normal colonic mucosa. (continued) ‘

 

 

53:22] Gene Title Tumor

1. Cell cyclg, cell growth and maintenance, apoptosis

LOC603 80 growth and transformation-dependent protein 0.4
Ceacaml carcinoembryonic antigen-related cell adhesion molecule 1 0.4
Txnip upregulated by 1,25-dihydroxyvitamin d-3 0.4
Pdgfa platelet derived growth factor, alpha 0.3
Pmp22 peripheral myelin protein 22 0.2
Cgrefl cell growth regulator with EF hand domain 1 0.1
II. Channel, transporters, & carriers

FabpS fatty acid binding protein 5, epidermal 8.9
Slc22a1 solute carrier family 22 (organic cation transporter), member 1 7.8
Rbpl retinol binding protein 1 7.6
Slc11a2 2:13;:33, grizelrlzmroton-coupled divalent metal ion 6.3
Slc7al $15112; 321:; :3in 7 (cationic amino acid transporter, y+ 5.4
S1c25a4 33:12:23: 3:11;); r2: (mitochondrial adenine nucleotide 4.6
--- apolipoprotein e 3.9
qul aquaporin l 3.6
--- hemoglobin beta chain complex 3.0
--- hemoglobin, alpha 1 2.7
Atp2cl atpase, ca++-sequestering 2.6
Abpl amiloride binding protein 1 2.2
Slc12a2 solute carrier family 12, member 2 2.1
Clnsla chloride channel, nucleotide-sensitive, 1A 2.1
Nup155 nucleoporin 155kd 2.0
--- transcobalamin 2 2.0
Atplal atpase, na+k+ transporting, alpha 1 0.5
Slclal solute carrier family 1, member 1 0.5
P2rx4 purinergic receptor p2x, ligand-gated ion channel, 4 0.5

 

74

 

 

TABLE 2. Genes significantly altered in AOM-induced colon cancers relative to

normal colonic mucosa. (continued) 1
Gene

 

symbol Gene Title Tumor

II. Channel, transporters, & carriers

--- similar to solute carrier family 37 (glycerol-3-phosphate 0.5

transporter), member 1 (loc294321), mrna

Slc5a6 solute carrier family 5, member 6 0.5
--- sulfotransferase family, cytosolic, 1c, member 2 0.4
--- chloride channel 2 0.4
Slc6a8 choline transporter 0.4
249858 plasmolipin 0.4
Slc16a1 solute carrier family 16, member 1 0.4
Slc5a3 solute carrier family 5 (inositol transporters), member 3 0.4
$ch Sal 0 211:; C(Larger:rntfiérrrrr1111:1215;mitochondrial carrier; dicarboxylate 0.4
--- atpase na+/k+ transporting beta 1 polypeptide 0.3
Atp2a3 atpase, ca++ transporting, ubiquitous 0.3
nyd4 fxyd domain-containing ion transport regulator 4 0.3
Slc13a2 solute carrier family 13, member 2 0.2
Atp12a atpase, h+/k+ transporting, nongastric, alpha polypeptide 0.1
111. Energy metabolism

Ass arginosuccinate synthetase 97
Argl arginase 1 l7
Hsdl 1b1 hydroxysteroid ll-beta dehydrogenase 1 3.8
Mel malic enzyme 1 3.5
Psatl phosphoserine aminotransferase 1 3.4
Scd2 stearoyl-coenzyme a desaturase 2 2.6
Oat ornithine aminotransferase 2.5
Pklr pyruvate kinase, liver and rbc 2.3
--- phosphofructokinase, platelet 2.2
des farensyl diphosphate synthase 2.2
Acsl4 fatty acid coenzyme a ligase, long chain 4 2.1
_anat acyl-coa:dihydroxyacetonephosphate acyltransferase 2.0

 

75

 

TABLE 2. Genes significantly altered in AOM-induced colon cancers relative to
normal colonic mucosa. (continued) 1

 

 

sfrfrlhbl Gene Title Tumor

111. Energy metabolism

Gyg glycogenin 2.0
Glul glutarnine synthetase 1 0.5
Pcca propionyl-coenzyme a carboxylase, alpha polypeptide 0.5
Acadm acetyl-coenzyme a dehydrogenase, medium chain 0.5
Pygb brain glycogen phosphorylase 0.5
Acads short chain acyl—coenzyme a dehydrogenase 0.4
Sord sorbitol dehydrogenase 0.4
I-Ikl hexokinase 1 0.4
Gpt glutamic-pyruvate transaminase (alanine aminotransferase) 0.4
—-- Peptide YY 0.3
--- aldolase B 0.3
ngcsZ 3-hydroxy-3-methylglutaryl-coenzyme a synthase 2 0.3
Eno3 enolase 3, beta 0.3
Pc pyruvate carboxylase 0.2
Hpgd nad-dependent 15-hydroxyprostaglandin dehydrogenase 0.1
--- phosphoenolpyruvate carboxykinase 1 0.0
IV. Extracellular matrix, tissur regeneration, wound repair

Dcn decorin 21
Apod apolipoprotein d 14
Lum lumican 12
C015a2 collagen, type V, alpha 2 9.9
Spare secreted acidic cysteine rich glycoprotein 9.4
--- fibronectin 1 8.5
Col3a1 collagen, type iii, alpha 1 5.0
Collal collagen, type 1, alpha 1 4.5
C0112a1 procollagen, type xii, alpha 1 4.1
Gpc2 glypican 2 (cerebroglycan) 3.9
Cyr61 cysteine rich protein 61 3.7

 

76

 

 

TABLE 2. Genes significantly altered in AOM-induced colon cancers relative to

normal colonic mucosa. (continued) ‘

 

 

85:12:] Gene Title Tumor

IV. Extracellular matrix, tissur regeneration, wound repair
Pgsg proteoglycan peptide core protein 3.6
Plod2 procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 3.6
Lamcl laminin, gamma 1 2.1
Cd81 CD 81 antigen 2.0
Tff3 trefoil factor 3 0.2
Dmbtl deleted in malignant brain tumors 1 0.1
V. Immung defense, imflammatogy response

rat anti-acetylcholine receptor antibody gene, rearranged i g
--- gamma-2a chain, vdjc region, complete eds 207
--- irnmunoglobulin heavy chain 1a (serum IgG2a) (predicted) 37
--- defensin ramp-3 precursor mrna, complete cds 29
--- gro 27
S100a9 leO calcium-binding protein a9 (calgranulin b) 26
S100a8 leO calcium-binding protein a8 (calgranulin a) 24
chr3 Fc receptor, IgG, low affinity III 19
--- anti-ngf30 antibody light-chain 16
Tacl tachykinin 1 15
Igha partial mrna for immunoglobulin alpha heavy chain (partial), 13

complete constant region
LOC60665 CXC chemokine LIX 12
Cls complement component 1, s subcomponent 11
--- similar to complement component 1, r subcomponent 1 1

(loc312705), mrna
lllb interleukin 1 beta 10
F2r coagulation factor II (thrombin) receptor 8.4
Illa interleukin 1 alpha 8.0
Aifl allograft inflammatory factor 1 7.2
--- MHC class II-associated invariant chain 6.9
Clqb complement component 1, q subcomponent, beta polypeptide 6.2

 

77

 

 

 

TABLE 2. Genes significantly altered in AOM-induced colon cancers relative to
normal colonic mucosa. (continued) 1

 

 

$313221 Gene Title Tumor

V. Immune, defense, imflarrTmatog response - l

Nos2 nitric oxide synthase 2, inducible 6.1
CxcllO chemokine (C-X-C motif) ligand 10 6.1
Pla2g2a phospholipase a2, group iia (platelets, synovial fluid) 6.1
--- ig germline kappa-chain c-region 6.1
F3 coagulation factor 3 5.8
Cd2 CD2 antigen 5.8
C0120 small inducible cytokine subfamily a20 5.0
Lyz lysozyme 4.3
RTl-Dbl RTl class II, locus Dbl 3.9
chr2b fc receptor, igg, low affinity iib 3.8
--- chemokine (c-c motif) ligand 2 3.8
RTl-Bb RTl class II, locus Bb 3.3
--- prostaglandin-endoperoxide synthase 2 3.2
Cxcr4 chemokine (C-X-C motif) receptor 4 3.1
--- RTl class II, locus Ba 2.8
Mfge8 milk fat globule-egf factor 8 protein 2.7
Mxl myxovirus (influenza virus) resistance 2.6
C d7 4 CD74 antigen (invariant polpypeptide of major histocompatibility 2. 6

class II antrgen—assocrated)

Il6r interleukin 6 receptor 2.5
RTl-Da RTl class II, locus Da 2.3
11er interleukin 1 receptor, type ii 2.2
Hla-dmb rtl class ii, locus dmb 2.0
Hla-dma rtl class ii, locus dma 2.0
Cd59 CD59 antigen 0.5
Cd14 CD14 antigen 0.5
chrt Fc receptor, IgG, alpha chain transporter 0.5
Tlr4 toll-like receptor 4 0.5

 

 

78

 

 

TABLE 2. Genes significantly altered in AOM-induced colon cancers relative to
normal colonic mucosa. (continued) I

 

 

sfrfrlbbl Gene Title Tumor

V. Immune_, defense, imflammatopy response

--- polymeric immunoglobulin receptor 0.3
1115 interleukin 15 0.3
1118 interleukin 18 0.2
Sfipd surfactant associated protein d 0.2
VI. Proteolysis and peptidolysis

Mmp7 matrix metalloproteinase 7 46
Plat plasminogen activator, tissue 19
Mmp12 matrix metalloproteinase 12 14
Mmp2 matrix metalloproteinase 2 (72 kda type iv collagenase) 13
Mmpl3 matrix metalloproteinase 13 12
Pace4 subtilisin - like endoprotease 9.1
Plau plasminogen activator, urokinase 4.4
Mmp3 matrix metalloproteinase 3 2.9
Ctsl cathepsin L 2.3
Ctse cathepsin E 2.3
Tpp2 tripeptidylpeptidase ii 2.1
Psm02 proteasome (prosome, macropain) 26s subunit, atpase 2 2.0
Ctss cathepsin S 0.5
Pcsk7 proprotein convertase subtilisin / kexin, type 7 0.5
Ctsd cathepsin D 0.4
Klks3 kallikrein, submaxillary gland $3 0.4
Dpp4 dipeptidylpeptidase 4 0.3
Naaladll n-acetylated alpha-linked acidic dipeptidase-like (ileal peptidase 0.2

1100)

Ngfg nerve grth factor, gamma 0.2
Mepla meprin 1 alpha 0.2

 

 

 

1 Data expressed as mean-fold change differences in tumor tissue relative to values for
non-neoplastic colon mucosa (n = 2-4/ group). A full listing of remaining transcripts
can be found in Appendix A.

79

 

Relative fold-change

 

 

 

 

 

Lysozyme DMBTl NP defensin sPLA2
NP defensin 3a

FIGURE 2. RT-PCR analysis of genes differentially expressed in
AOM-induced colon cancers. Values represent relative fold-changes
in RNA of tumor tissue normalized to non-neoplastic mucosa.
Abbreviations: Deleted in malignant brain tumors 1 (DMBTI), group

IIA secretory phospholipase A2 (sPLA2).

80

E, DISCUSSION.

Chronic inflammation has long been recognized to be involved in the etiology of
colon cancer, and clinical, epidemiological, and experimental studies have established the
efficacy of non-steroidal anti-inflammatory drugs (NSAIDs) in reducing the occurrence
and mortality from CRC (9, 23). In this study, the most highly induced class of genes
affected in colon tumors were those involved in inflammation and immunity with
evidence of infiltration of lymphocytes, macrophages, neutrophils along with tissue
damage and attempts at repair. For example up-regulation of COX-2, sPLA2, and iNOS,
have been described previously in carcinogen-induced cancers as well as in human
cancers (20-22, 24-25). The two former produce lipid mediators that stimulate cell
proliferation and angiogenesis and inhibit apoptosis (26-29) whereas nitric oxide has
been implicated in DNA damage and inactivation of tumor suppressor proteins through
post-translational modification (30). S100 A and B are of macrophage and neutrophil
origin and serum levels increase acutely during inflammation (31). S100 proteins form a
lipid-scavenging complex within the cytosol of leukocytes and are thought to be a latent
source of arachidonic acid for target cells at sites of inflammation (31). Also highly
induced in AOM-induced colon cancers was the innate defense gene NP defensin 30:. NP
defensin 3a is a cationic peptide induced during inflammation that exhibits a range of
anti-microbial activity in vitro (32). Melle et al. (33) recently reported potential
usefulness of NP defensin 30 as a prognostic marker for recognition of individuals with
colorectal cancer with a serum protein sensitivity of 69% and specificity of 100%.

In contrast to the number of inflammatory genes induced in colon cancers, we

detected a lower expression of IL-18 and IL-15. IL-18 induces release of the Th1

81

 

cytokine interferon (IFN)-y, which facilitates tumor cell recognition by stimulating
cytotoxicity of natural killer (NK) cells, T lymphocytes, and macrophages and enhancing
major histocompatability class (MHC) expression and antigen processing (34-35). IL-18
mRNA is depressed in human colon cancer tissue and absence of IL-18 is associated with
poor prognosis including lymph node and liver metastasis (35). Similarly, IL-15
augments innate and adaptive immune responses by activating NK cell activity and
stimulating CD8+ T cell recognition of tumor cells (36). A lower expression of IL-18
and IL-15 in AOM-induced tumors is suggestive of host evasion from immune
surveillance and potential for metastatic capability.

Alterations in extracellular matrix (ECM) components are commonly observed
during colon carcinogenesis and serve as a reservoir for grth factors, facilitate
migration and proliferation, as well as contribute to angiogenesis (37). In this study
AOM-induced colon cancers displayed higher transcripts for the ECM components
decorin, fibronectin 1, and collagen lotl. Decorin is a proteoglycan characteristic of
stromal tissue (37) that is transcriptionally activated by hypomethylation in human colon
cancer tissue (38). Decorin binds to and can inhibit transforming growth factor B
(TGFB), which normally exerts inhibitory effects on colonocyte proliferation (39).
Fibronectin and collagen lorl have been implicated in a variety of processes including
cell adhesion, migration, and chemotaxis. Both genes are increased in colon cancers and
other hyperproliferative orders, and epithelial transcription decreases during terminal
differentiation (40-43). Pleiotrophin, a heparin-binding cytokine was also highly

expressed in AOM-induced colon cancer (44). The gene encoding pleiotrophin is

82

 

increased at active sites of neovasculargenesis including during development, ischemia,
and in several cancers (44).

A high percentage of genes involved in the cell cycle were also affected in AOM-
induced cancers. For example, tumors displayed a lower expression of the tumor
suppressor gene APC and higher expression of the cell cycle regulators CDK4, cyclin E,
and cyclin D1. APC mutations are common and permissive for human colon cancer
development, but not frequently observed in AOM-induced colon cancers (17). APC
expression increases in normal colon tissue as cells migrate up the colon crypt, and
introduction of APC into cancer cells initiates apoptosis (45). Lower expression of APC
in this study is likely a result of altered differentiation in colon tumors concomitant with
evasion of apoptosis. AOM-induced tumors also displayed a lower expression of the
glycoproteins CEACAM l and DMBTl. CEACAMl was identified as a putative tumor
suppressor gene involved in inducing apoptosis in response to hyperplasia (46). Nikkita
et al. (46) reported loss of CEACAM 1 is occurs with a similar frequency in hyperplastic
polyps and colon cancers, suggesting it precedes defects in the APC pathway. Loss of
DMBTl has not been previously reported in AOM colon cancers, but has been detected
in human colon (47), neuroblastomas (48), and lung carcinomas (49), suggesting a
possible tumor suppressive role. The firnction of DMBTl has not been fully established,
but has been implicated as a scavenger receptor involved in epithelial defense against
bacteria (50).

Among genes most underrepresented in AOM-induced colon cancers included ion
transporters and metabolic enzymes. The almost complete absence of some of the genes

encoding enzymes involved in fatty acid oxidation and electron transport as well as in

83

gluconeogenesis, implies generalized defects in mitochondrial function and energy
metabolism. Normal colon tissue preferentially oxidizes the short-chain fatty acid
butyrate as an energy source (51). During cancer progression, a switch to glycolytic
metabolism has been reported and thought to be a consequence of hypoxia in tumor tissue
(52). A lower abundance of mitochondrial enzymes also been recently described in
human cancers (53), and may explain the positive association of insulin resistance and
hyperglycemia on CRC development, more so for advanced stages (54-56). Other
metabolic enzymes including arginosuccinate synthase and arginase as well as cationic
amino acid transporters were highly induced in colon cancers, potentially contributing to
epithelial production of substrates for nitric oxide and polyamine synthesis, respectively.
In conclusion, this research provided more insight into global gene alterations
contributing to AOM-induced colon carcinogenesis. A majority of genes induced during
cancer progression are related to inflammatory and immune responses and angiogenesis
with evidence of lower production of cytokines involved in anti-tumor immunity. Genes
involved in extracellular matrix formation were also highly represented. A lower
expression of ion transporters and metabolic enzymes highlights cellular disturbances in

ion and water absorption and mitochondrial metabolism during cancer progression.

84

_13,

10.

11.

12.

13.

LITERATURE CITED.

Fearon ER and Vogelstein B. (1990) A Genetic Model for Colorectal
Tumorigenesis. Cell. 61(5): 759-767.

Kinzler KW and Vogelstein B. (1996) Lessons from hereditary colorectal cancer.
Cell. 87(2): 159-170.

Ilyas M, Straub J, Tomlinson [PM and Bodmer WF. (1999) Genetic pathways in
colorectal and other cancers. European Journal of Cancer. 35(3): 335-351.

Williams NS, Gaynor RB, Scoggin S et al. (2003) Identification and validation of
genes involved in the pathogenesis of colorectal cancer using cDNA microarrays
and RNA interference. Clinical Cancer Research. 9(3): 931-946.

Bertucci F, Salas S, Eysteries S et al. (2004) Gene expression profiling of colon
cancer by DNA microarrays and correlation with histoclinical parameters.
Oncogene. 23(7): 1377-1391.

Barrier A, Lemoine A, Boelle PY et al. (2005) Colon cancer prognosis prediction
by gene expression profiling. Oncogene. 24(40): 6155-6164.

Shih W, Chetty R and Tsao MS. (2005) Expression profiling by microarrays in
colorectal cancer (Review). Oncology Reports. 13(3): 517-524.

Rhodes JM and Campbell BJ. (2002) lnflarnmation and colorectal cancer: IBD-
associated and sporadic cancer compared. Trends in Molecular Medicine. 8(1): 10-
16.

Herendeen JM and Lindley C. (2003) Use of NSAIDs for the chemoprevention of
colorectal cancer. Annals of Pharmacotherapy. 37(11): 1664-1674.

American Cancer Society. (2005) Cancer Facts & Figures 2005.
http://www.cancer.org/downloads/STT/CAFF2005f4PWSecure®df

Pories SE, Ramchurren N, Summerhayes I and Steele G. (1993) Animal models for
colon carcinogenesis. Archives of Surgery. 128: 647-53.

Reddy BS. (2004) Studies with the azoxymethane-rat preclinical model for
assessing colon tumor development and chemoprevention. Environmental and
molecular mutagenesis. 44 (1): 26-35.

Druckrey H, Preussman R, Matzkies F and Ivankovic S. (1967) Erzeugung von

Darmkrebs bei Ratten durch 1,2-dimethylhydrazin. Naturewissenschaften. 54: 285-
286, 1967.

85

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Druckrey H. (1968) Organotropic carcinogenesis and mechanisms of action of
symmetrical dialkylhydrazines and azo-and azoalkanes. Food Cosmet. T oxicol. 6:
578-579.

Druckery H. (1970) Production of colonic carcinomas by 1,2-dialkylhydrazines and
azoalkanes. In Carcinoma of the colon and antecedent epithelium, WJ Burdette, ed.
Springfield, Charles C Thomas; pp. 267-79.

Takahashi M and Wakabayashi K. (2004) Gene mutations and altered gene
expression in azoxymethane-induced colon carcinogenesis in rodents. Cancer
Science. 95(6): 475-480.

Cademi G, De Filippo C, Luceri C et al. (1997) Apc mutations in aberrant crypt
foci (ACF) and colonic tumors induced by azoxymethane in F344 rats. Proc. Amer.
Assoc. Cancer Res. 38: 467.

Takahashi M, Fukuda K, Sugimura T and Wakabayashi K. (1998) Beta-catenin is
frequently mutated and demonstrates altered cellular location in azoxymethane-
induced rat colon tumors. Cancer Research. 58(1): 42-46.

Vivona AA, Shpitz B, Medline A et al. (1993) K-Ras Mutations in Aberrant Crypt
Foci, Adenomas and Adenocarcinomas During Azoxymethane-Induced Colon
Carcinogenesis. Carcinogenesis. 14(9): 1777-1781.

DuBois RN, Radhika A, Reddy BS and Entingh AJ. (1996) Increased
cyclooxygenase-2 levels in carcinogen-induced rat colonic tumors.
Gastroenterology. 1 10(4): 1259-1262.

Watanabe K, Kawamori T, Nakatsugi S and Wakabayashi K. (2000) COX-2 and
iNOS, good targets for chemoprevention of colon cancer. Biofactors. 12(1-4): 129-
133.

Ohta T, Takahashib M, and Ochiaic A. (2005) Increased protein expression of both
inducible nitric oxide synthase and cyclooxygenase-2 in human colon cancers.
Cancer Letters. 1-8 (epub ahead of print).

Reddy BS, Rao CV and Seibert K. (1996) Evaluation of cyclooxygenase-2 inhibitor
for potential Chemopreventive properties in colon carcinogenesis. Cancer Research.
56(20): 4566-4569.

Yarnashita S, Ogawa M, Sakamoto K et al. (1994) Elevation of Serum Group-Ii

Phospholipase A(2) Levels in Patients with Advanced Cancer. Clinica Chimica
Acta. 228(2): 91-99.

86

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

Ikegami T, Matsuzaki Y, Shoda J et al. (1998) The Chemopreventive role of
ursodeoxycholic acid in azoxymethane-treated rats: suppressive effects on

enhanced group II phospholipase A(2) expression in colonic tissue. Cancer Letters.
134(2): 129-139.

Bingham CO and Austen KF. (1999) Phospholipase A(2) enzymes in eicosanoid
generation. Proceedings of the Association of American Physicians. 111(6): 516-
524.

Ashktorab H, Dawkins FW, Mohamed R et al. (2005) Apoptosis induced by aspirin
and 5-fluorouracil in hrunan colonic adenocarcinoma cells. Digestive Diseases and
Sciences. 50(6): 1025-1032.

Tarnawski AS and Jones MK. (2003) Inhibition of angiogenesis by NSAIDs:
molecular mechanisms and clinical implications. Journal of Molecular Medicine-
Jmm. 81(10): 627-636.

Williams CS, Mann M and DuBois RN. (1999) The role of cyclooxygenases in
inflammation, cancer, and development. Oncogene. 18(55): 7908-7916.

Pavlick KP, Laroux S, Fuseler J et al. (2002) Role of reactive metabolites of

oxygen and nitrogen in inflammatory bowel disease. Free Radical Biology and
Medicine. 33(3): 311-22.

Nacken W, Roth J, Sorg C and Kerkhoff C. (2003) SlOOA9/S100A8: Myeloid
Representatives of the S100 Protein Family as Prominent Players in Innate
Immunity. Microsc. Res. Tech. 60: 569—580.

Ganz T and Lehrer RI. (1994) Defensins. Current Opinion in Immunology. 6(4):
584-589.

Melle C, Ernst G, Schimmel B et a1 (2005) Discovery and Identification of a-
Defensins as Low Abundant, Tumor-Derived Serum Markers in Colorectal Cancer.
Gastroenterology. 129(1): 66-73.

Tartour E and Fridman WH. (1998) Cytokines and cancer. Int. Rev. Immunol. 16:
683-704.

Pages F, Bergerb A, Lebel-Binaya S et al. (2000) Proinflammatory and antitumor
properties of interleukin-18 in the gastrointestinal tract. Immunology Letters. 75(1):
9-14

Araki A, Hazama S, Yoshimura K et al. (2004) Tumor secreting high levels of IL-

15 induces specific immunity to low immunogenic colon adenocarcinoma via
CD8+ T cells. Int J Mol Med. 14(4): 571-6.

87

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

Wemert N. (1997) The multiple roles of tumour stroma. Virchows Archiv-an
International Journal of Pathology. 430(6): 433-443.

10220 RV. (1995) Tumor Stroma as a Regulator of Neoplastic Behavior - Agonistic
and Antagonistic Elements Embedded in the Same Connective-Tissue. Laboratory
Investigation. 73(2): 157-160.

Massague J. (2000) How cells read TGF-beta signals. Nature Reviews Molecular
Cell Biology. 1 (3): 169-178.

Gradl D, Kuhl M and Wedlich D. (1999) The Wnt/W g signal transducer beta-
catenin controls fibronectin expression. Molecular and Cellular Biology; 19(8):
5576-5587.

Takayasu H, Horie H, Hiyama E et al. (2001) Frequent deletions and mutations of
the beta-catenin gene are associated with overexpression of cyclin D1 and
fibronectin and poorly differentiated histology in childhood hepatoblastoma.
Clinical Cancer Research. 7(4): 901-908.

deGiorgio-Miller AM, Trehame LJ, McAnulty RJ et al. (2005) Procoltagen type I
gene expression and cell proliferation are increased in lipoderrnatoscterosis. British
Journal of Dermatology. 152(2): 242-249.

Vachon PH, Simoneau A, Herringgillam FE and Beaulieu JF. (1995) Cellular
Fibronectin Expression Is Down-Regulated at the Messenger-RNA Level in

Differentiating Human Intestinal Epithelial-Cells. Experimental Cell Research.
216(1): 30-34.

Christrnan KL, Fang Q, Kim AJ et al. (2005) Pleiotrophin induces formation of
functional neovasculature in vivo. Biochem Biophys Res Commun. 332(4): 1146-
52.

Morin PJ, Vogelstein B, Kinzler KW. (1996) Apoptosis and APC in colorectal
tumorigenesis. Proc Natl Acad Sci U S A. 93(15):7950-4.

Nittka S, Gunther J, Ebisch C, et al. (2004) The human tumor suppressor
CEACAM] modulates apoptosis and is implicated in early colorectal
tumorigenesis. Oncogene. 23(58): 9306-9313.

Mori M, Shiraishi T, Tanaka S et al. (1999) Lack of DMBTl expression in
oesophageal, gastric and colon cancers. British Journal of Cancer. 79(2): 211-213.

Munoz J, Lazcoz P, Inda MD et al. (2004) Homozygous deletion and expression of

PTEN and DMBTl in human primary neuroblastoma and cell lines. International
Journal of Cancer. 109(5): 673-679.

88

49.

50.

51.

52.

53.

54.

55.

56.

Wu W, Kemp BL, Proctor ML et al. (1999) Expression of DMBTI, a Candidate

Tumor Suppressor Gene, Is Frequently Lost in Lung Cancer. Cancer Research.
59(8): 1546-51.

Kang WQ and Reid KBM. (2003) DMBTl, a regulator of mucosal homeostasis
through the linking of mucosal defense and regeneration? Febs Letters. 540(1-3):
21-25.

Roediger WE and Truelove SC. (1979) Method of preparing isolated colonic
epithelial cells (colonocytes) for metabolic studies. Gut. 20(6): 484-8.

Dang CV and Semenza GL. (1999) Oncogenic alterations of metabolism. Trends in
Biochemical Sciences. 24(2): 68-72.

Birkenkamp-Demtroder K, Christensen LL, Olesen SH et al. (2002) Gene
expression in colorectal cancer. Cancer Research. 62: 4352-4363.

Chang CK and Ulrich CM. (2003) Hyperinsulinaemia and hyperglycaemia:
possible risk factors of colorectal cancer among diabetic patients. Diabetologia.
46(5): 595-607.

Komninou D, Ayonote A, Richie JP and Rigas B. (2003) Insulin resistance and its
contribution to colon carcinogenesis. Experimental Biology and Medicine. 228(4):
396-405.

Kaaks R, Toniolo P, Akhmedkhanov A et al. (2000) Serum C-peptide, insulin-like

growth factor (IGF)-I, IGF-binding proteins, and colorectal cancer risk in women.
Journal of the National Cancer Institute. 92(19): 1592-1600.

89

CHAPTER IV.

MICROARRAY ANALYSES OF GENES DIFFERENTIALLY EXPRESSED BY

DIET (BLACK BEANS AND SOY FLOUR) DURING AOM-INDUCED COLON

CARCINOGENESIS IN RATS

9O

A, ABSTRACT.

We have previously demonstrated that feeding diets containing beans (black
beans or soy flour) inhibits azoxymethane (AOM)-induced colon cancer in rats. The
objective of this study was to identify genes altered during the early stages of AOM-
induced colon carcinogenesis as well as candidate genes potentially involved in
chemoprevention by beans. Ninety-five male Fischer F344 rats were obtained at 3
weeks of age and fed control (AIN-93G) diets upon arrival. At 4 and 5 weeks of age, rats
were injected with AOM (15 mg/kg, n=75) or saline (SHAM, n=20) and at 6 weeks of
age were separated into 3 groups and fed either the control (AIN), black bean (BB), or a
soy flour (SF)-based diet. Rats were sacrificed at 36 weeks of age, and microarrays were
performed on mRNA isolated from normal-appearing distal colonic mucosa. In animals
treated with AOM, there was higher abundance of genes involved in immune function,
including several MHC II-associated antigens and the antimicrobial genes lysozyme, NP
defensin 30, and sPLA2. BB- and SF-fed rats exhibited a higher expression of genes
involved in energy metabolism and a lower expression of inflammatory (sPLA2, NP
defensin 3a) and cell cycle-associated (cyclin B1, CDC2, TOP2A, GADD450L) genes.

Genes involved in the extracellular matrix (collagen lal, fibronectin 1) and in innate

immunity (NP defensin 3a, sPLA2) were induced by AOM injections in all diets, but
were lowest in bean-fed animals. The gene change profile suggests that bean diets inhibit
tumorigenesis by enhancing cellular differentiation and reducing early inflammatory

events in non-neoplastic colonic mucosa.

91

1_3_._ INTRODUCTION.

Colorectal cancer (CRC) is currently the fourth most common cancer in the
United States and the second leading cause of cancer mortality. An estimated 145, 290
new cases of CRC will be diagnosed alone in the US in 2005 (1). Although the
development of CRC is likely multi-factorial, there is reason to believe that a majority of
sporadic CRC cases can be substantially reduced, if not prevented, through modification
of diet (2, 3). Among dietary factors associated with a decrease risk, epidemiological
studies indicate that populations consuming higher intakes of legumes (peas, beans,
lentils, peanuts) have a lower occurrence of (4-6) and mortality from CRC (7).
Experiments conducted in our laboratory (8-12) and by others (13-14) have also
demonstrated the potential of bean-based diets to inhibit carcinogen-induced colon cancer
in rodents. For example, Hughes et al. (13) and Hangen and Bennink (12) found that rats
fed dry beans (pinto, navy, or black beans) had a 50-57% lower incidence of colon cancer
than rats fed a casein-based diet. Similarly, in a series of experiments, Bennink et al. (8-
11) reported a significant reduction in colon tumor incidence and tumor burden in rats fed
defatted soy flour (43%, 0.67 tumors/animal) compared to either ethanol-extracted soy
concentrate (68%, 1.1 tumor/animal) or casein (68%, 1.43 tumor/animal). Several of the
mechanisms proposed to account for the anti-tumorigenic effect of legumes have been
attributed to the presence of one or more bioactive compounds (ie. phytoestrogens,
protease inhibitors, phytic acid, saponins, and phytosterols), some of which inhibit one or
more steps involved in cancer development (15-27). Although important, there is

currently insufficient understanding of the cellular and molecular basis underlying colon

92

cancer inhibition by beans and the relative contribution of some of these compounds to
the observed anti-neoplastic effects in vivo.

The azoxymethane (AOM)-treated rodent model has been utilized extensively to
examine dietary influences on colon cancer, and many of the common genetic and
pathogenic changes contributing to human colon cancers have also been observed during
AOM-induced colon carcinogenesis (28-32). The purpose of this study was to provide
insight into the cellular mechanisms contributing to colon cancer inhibition by beans in
vivo by profiling changes in gene expression. Microarrays were performed on mRNA
isolated from distal colonic epithelium of saline and AOM-injected F344 rats fed either a
casein (AIN), black bean (BB), or defatted soy flour (SF) diet for 31 weeks. We chose to
focus on the distal segment because most tumors develop in this area using standard
protocol (15 mg/kg AOM) and there is evidence for site-specific effects of food
constituents on tumorigenesis (11, 33). It was anticipated that genes most important to
dietary suppression of tumorigenesis would be similarly affected by black beans and soy
flour and have altered expression (increased or decreased) that corroborated tumor

incidence.

Q_. MATERIALS AND METHODS.
Animal care and experimental diets.

This study was conducted in conformity with the regulatory guidelines of the
Michigan State University Committee on Animal Use and Care. Ninety-five male
Fischer (F344) rats were obtained from Harlan Sprague-Dawley (Indianapolis, IL) at 3

weeks of age and housed in plastic cages (2-3 rats/cage) in temperature (23°C 2!: 2°) and

93

humidity (40-60%) controlled rooms with a 12 hour light/dark cycle. Throughout the
experiment animals had free access to food and distilled water and were assessed daily
for health status and monthly for weight gain.

One control and two experimental diets were formulated based on the AIN-93G
rodent diet with modifications (34), to contain the majority (ie >85%) of protein from
either 1) high nitrogen casein (AIN), 2) black beans (BB), or 3) defatted soy flour (SF)
(Archer Daniels Midland; Decantur, IL) and matched to have similar nutrientzenergy
ratios (TABLE 1). Black beans were soaked overnight in distilled water, conventionally
cooked for 20-30 minutes, dried at 58 °C, and then finely ground through a 1.6 mm
diameter mesh sieve prior to mixing with other diet ingredients. All diets contained
approximately 18.9% (wt/wt) total protein, 11.3% dietary fiber, and 16.7% fat (wt/wt).
Casein and tryptophan were added to black bean diet and methionine was added to all
diets to increase the amino acid score. Lard and soybean oil were used as the fatty acid
source, adjusted so the PUFA:MUFA:SFA ratios were 0.86:1 .0:0.80 respectively.
Experimental design.

The design for this experiment was a 3X2 factorial with 3 dietary treatments
(AIN, BB, SF) and 2 injection regimes (AOM, SHAM). Upon arrival, animals were fed
the control (AIN) diet and allowed one week to acclimatize to new conditions. At 4 and
5 weeks of age, rats received subcutaneous injections (100 uL) of either 15 mg/kg of
azoxymethane (AOM) prepared in saline (n=75; Ash Stevens, Detroit, M1) or saline

(SHAM, n=20). Animals were fed the control (AIN) diet until one week after the final

94

 

TABLE 1. Nutrient composition of experimental diets.

 

 

 

g/Ioogdiet

Medient AIN Black Bean Soy
Casein 20 2.7 --
Dried, ground black beans -- 74 --
Defatted soy flour (55% protein) -- -- 34
Cornstarch 45 -- 36
Sucrose 1.8 1.8 1.8
(Oil in diet) (--) (1 .1) (0.27)
Added soy oil 5.2 6.6 6.6
Corn oil 1.7 -- --
Lard 9.7 9.1 9.8
(Fiber in diet) (--) (11) (5.0)
Added fiber (cellulose) 11 -- 5.9
Mineral mix 3.9 3.9 3.9
Vitaminmix 1.1 1.1 1.1
Methionine 0.33 0.40 0.33
Tryptophan -- 0.004 --
Calcium carbonate 0.25 0.25 0.25
Choline bitartrate 0.28 0.28 0.28
Tert-butylhydroquinone 0.002 0.002 0.002
Totals: 100 100 100

 

95

 

injection, when they were randomized by weight to either continue on the control (AIN)
diet or to be fed one of the experimental diets (BB or SF). At 36 weeks of age, animals
were sacrificed by C02 inhalation and exsanguination, and the colon was immediately
excised, opened longitudinally, and rinsed briefly in tap water to remove debris.
Macroscopic tumors, when present, were excised and stored at —80 °C to be analyzed as a
separate part of this study (CHAPTER III). The colon was then transected into
proximal and distal segments and epithelial cells were collected by gently scraping
normal-appearing mucosa from the distal half of the colon (excluding the lowermost 1
cm) with a glass slide. Samples were snap frozen and stored at —80 °C until RNA
extraction could be performed.

Microarray target preparation and hybridization.

Affymetrix RU34A rat genome chips (Santa Clara, CA) were used in this
experiment. For total RNA isolation, < 100 mg distal colonic mucosa was homogenized
using a Tekmar homogenizer in one mL of TRIzol reagent containing 2 uL of RNase-free
glycogen according to the manufacturers instructions (Gibco, Carlsbad, CA). After RNA
extraction, samples were cleaned with RNeasy mini columns (Qiagen, Valencia, CA),
quantified using a UV spectrophotometer (Azoo/Azgo), and the quality of RNA assessed by
agarose-formaldehyde gel electrophoresis. Only high quality RNA was used in
subsequent steps.

Biotinylated cRNA was prepared in accordance with instructions supplied in the
GeneChip Expression Manual (Affymetrix, Santa Clara, CA). Double-stranded cDNA
was synthesized from 10 pg of total RNA, pooled from 2-4 animals/treatment, using T7-

(dT)24 primers containing a T7 RNA polymerase promoter site (Proligo, Boulder, CO)

96

and the Superscript II system (Invitrogen, Carlsbad, California). Biotinylated cRNA was
prepared using the Enzo BioArray Higtheld RNA Transcript Labeling Kit (Affymetrix,
Santa Clara, CA) and then purified with RNeasy mini columns. Approximately 15 pg
cRNA was fragmented at 94 °C for 35 minutes and hybridized to RGU34A rat genome
chips for 16 hours at 45 °C. Following hybridization, arrays were washed and stained

with a streptavidin-phycoerythrin conjugate on an Affymetrix Fluidics station according
to standard protocol. Processed arrays were scanned at 570 nm using a Hewlett Packard

GeneArray Scanner.
Quantitative reverse transcription-PCR (R T -PCR) analysis.

The pooled RNA samples used for oligonucleotide microarrays were also used to
confirm gene changes with RT-PCR. Gene specific primers for cell division cycle 2
(CDC2), cyclin B1, topoisomerase 2A (TOP2A), grth arrest and DNA-damage
induced 450i (GADD450L), and group IIa secretory phospholipase A2 (sPLA2) were
designed with the Primer Express 2.0 program (Applied Biosystems, Foster City, CA).
The sequences of the primer pairs used are shown in TABLE 2. Single-stranded (85)
cDNA was synthesized from 2.5 pg of total RNA using T7-(dT)24 primers (Proligo,
Boulder, CO) and the Superscript II system (Invitrogen, Carlsbad, CA). Reverse
transcription was performed in a thermocycler following the Superscript first strand
synthesis protocol (Invitrogen, Carlsbad, California) with the following modifications:
temperature of reaction was cooled to 20 °C for 5 minutes following annealing of primer
and 53 cDNA synthesis time was increased to 60 minutes for 42 °C. Following the
reaction, ss cDNA was isolated with phase lock gel (Eppendorf), precipitated overnight,

and the pellet was resuspended in ddeO to a working concentration of 10 ng/pL.

97

 

A0300 8 880000800 0228... .8 0020 0:0 805800 000 08005
000000.820 05. 0005 5380 800000 <0 00005883 800000 0 20.0 80000 :00 H3008050.... _

 

80000080050500.8000 005800000000800000 08000
8000800000508005 8080800000000000 004080
008000<0080000<0550 805800080800000000 80000
08008080005000.000808 80800000850000 8 00000
0.8000008000000008 80000000000500.0000 0000
008000080008055 805005808000008 008-0
00-00 .28.. 000.50 00.0 SEE 0.0.00... 2.00

 

WHY—rhy— 08 000: 0.80.— 0008..."— .N mam—<8

 

98

Quantitative determination of gene expression was performed with the ABI
Prism7000 (Perkin Elmer Corp., Foster City, CA) using the Syber Green Universal
Master Mix (Applied Biosystems, Foster City, CA). The reaction mixture (25 pL total
volume) contained 20 ng ss cDNA, 12.5 pL SYBR Green RT-PCR Master mix, and 10.5
pL of diluted primers (forward and reverse). The final concentration (per 25 pL reaction
volume) of primers was as follows: 600 nM for TOP2A and cyclin BI, and 1.2 pM for
sPLA2, CDC2, GADD450L, and B-Actin. The real-time cycle conditions were as follows:
PCR initial activation step at 95 °C for 15 min and a total of 40 cycles for melting (95 °C,
15 s) and annealing/extension (60 °C, 1 min). All assays were performed in duplicates,
using 2-3 samples per group and relative fold-changes were quantified by using the
comparative CT (AACT) method (Applied Biosystems, User Bulletin #2) with the
AIN(saline-inj ected) group as the calibrator.

Statistical analysis methods.

Data for weight gain, microarrays, and RT-PCR were analyzed using the General
Linear Models procedure of SAS (SAS Institute, Cary, NC, Version 7.0). When
statistical differences were detected with the F statistic, individual comparisons were
made using the least significant difference (LSD) method. Tumor incidence data was
analyzed with a x2 test using the Proc Freq procedure in SAS.

Prior to statistical analyses fluorescence intensity data from microarrays were
globally scaled to a target intensity of 500 in Affymetrix Microarray Suite, version 5.0 to
control within-chip variations. Globally scaled data were then imported into GeneSpring
(Silicon Genetics, Inc., Redwood City, CA, Version 6.0) for normalization and filtering.

All chips were normalized to the median intensity of a set of invariant genes whose

99

expression across all conditions (injection type, diet) after global scaling showed less
than a 30% coefficient of variation (CV). A filtering step excluded genes not considered
“Present” or “Marginal” in at least 42% (10 of 24) of the samples. This step reduced the
number of genes to be analyzed from 8799 to 3931 genes (45% of genes present on the
chip). An additional filtering step limited the genes further to those exhibiting greater
than 1.3 or less than 0.7 fold-change difference between diets or injection type.
Normalized expression values were analyzed using the GLM procedure of SAS. When
present, duplicate transcripts were averaged following statistical analysis.

Differentially expressed transcripts (P<0.05) were broadly grouped into categories
based on known gene ontologies and biological functions reported in the literature. Gene
ontologies were retrieved using the Affymetrix NetAffix Analysis Center
(http://www.affvmetrix.com/angljsis/indexaffx) and the Rat Genome Database (RGD)
gene annotation tool (Rat Genome Database Web Site, Medical College of Wisconsin,
Milwaukee, Wisconsin; World Wide Web: http://rgd.mcw.edu/gatool/). For brevity,
only transcripts with known identities are shown. Data are presented as mean fold-
change differences standardized to the AIN (control) group for diet-dependent differences

or to the saline-injected group for inj ection-dependent changes.

g RESULTS.
Weight gain and tumor incidence.

There were no significant main effects of diet (AIN, BB, SF) or injection regime
(AOM vs saline) on body weight gain. The total weight gain (g, LSM i SEM) of rats

While on experimental diets was: AIN = 304 i 7.4, BB = 287 i 8.3, SF = 299 i 8.2.

100

There was a significant effect of diet on tumor incidence (P = 0.03). As previously
established, bean-based diets inhibited tumor incidence by ~60% compared to rats fed the
control diet. Tumor incidence was 75%, 33%, and 25% for rats fed the AIN, BB, or SF
diets, respectively (P < 0.05).

Microarray analysis.

Among the 8799 genes and ESTs present on the rat genome UG34A array, 316
transcripts passed the multiple filtering criteria employed to be further analyzed for
injection- and 928 for diet-dependent changes (see under statistical analyses). A total of
157 transcripts were significantly affected by injection regime (AOM vs. saline), 257 by
dietary treatment (AIN, BB, SF), and 9 were common to both lists (P < 0.05).
Transcripts differentially expressed were broadly classified into one of eleven functional
categories (FIGURE 1) and included:

(1) Cell cycle, cell growth & maintenance, apoptosis

(2) Channel, transporter, carrier proteins

(3) Electron transfer, oxidoreductase, detoxification

(4) Energy metabolism

(5) Extracellular matrix, cell adhesion, cytoskeleton

(6) hnmune, defense, stress

(7) Intracellular protein metabolism

(8) Nucleic acid binding, transcription regulation

(9) Signal transduction

(10) Enzymes, other

(11) Other

101

, El AOM Treatment

Cell cycle, cell growth and maintenance, I Dietary Treatment

apoptosis

Channel, transporter, carrier a

Electron tranport, oxidoreductase,
detoxification

Energy Metabolism

Enzyme, other

Extracellular rmtrix, cell adhesion,
cytoskeleton

 

Immune, defense, inflammation, stress

Intracellular protein metabolism

Nucleic acid binding, tramcripu'on
regubtion

 

Signal transduction

 

Other

w W

l
l
r

 

0% 5% 10% 15% 20%
Percentage of known transcripts

FIGURE 1. Biological classification of known transcripts significantly
altered in the distal colonic epithelium of male F344 rats as affected by

AOM-injections and dietary (AIN, BB, SF) treatment (P < 0.05).

102

Genes differentially expressed by carcinogen (A OM) injection.

There was a greater mRNA abundance for 108 transcripts and lower abundance
for 48 transcripts in the colon of rats injected with carcinogen (AOM) as compared to
saline-injected controls (SHAMs). Of these genes, 112 (75 up-regulated, 37 down-
regulated) were categorized according to biological functions and are presented. in
FIGURE 2. As shown, a majority of known transcripts affected (59%) are associated
with immune, defense, stress (n=17, 15%), signal transduction (n=l6, 14%), other (n=15,
13%), or intracellular protein metabolism (n=13, 12%).

Genes involved in antigen presentation (CD74), anti-microbial proteins (NP
defensin 3a, lysozyme), inflammatory proteins (sPLA2), and extracellular matrix
proteins (collagen 10:1, fibronectin l) were among the genes most highly induced (>1.5-
fold) by AOM-treatment (TABLE 3). Several of these genes have previously been
shown to be over-expressed in carcinogen-induced colon cancer (35) and inflammatory
conditions of the colon (36). Genes involved in ribosomal protein synthesis, including
components of the 40S ribosomal protein subunit (s7, s4, s9) and the 60S unit (137, 14,
136a) were also moderately induced by carcinogen injections. A full listing of the
remaining transcripts significantly affected by AOM treatment is available in the

APPENDICES as SUPPLEMENTARY TABLE 2.

103

 

 

TABLE 3. Select genes differentially expressed by carcinogen (AOM) treatment
in the distal colonic epithelium of male F344 rats. '

Saline- AOM-
Gene symbol Common Injected Injected

I. Extracellular matrix, cell adhesion, cytoskeleton

 

Collal collagen, type 1, alpha 1 1.0 a 1.8 b
Fnl fibronectin 1 1.0 ‘ 1.7 b
Vim vimentin 1.0 a 1.5 b
TmsblO thymosin, beta 10 1.0 a 1.3 b
Col3a1 collagen, type iii, alpha 1 1.0 a 1.3 b
CeacamlO c-cam4 protein 1.0 a 1.3 b
Cdc42bpb cdc42-binding protein kinase beta 1.0 b 0.6 a
II. Immune defense inflammation stress

---- similar to 1G kappa-chain V-V region K2 precursor 1.0 a 3.3 b
Pla2g2a phospholipase a2, group iia 1.0 a 2.4 b
Lyz lysozyme 1.0 a 2.1 b
RTl-Dbl rtl class ii, locus dbl 1.0 a 1.8 b
C d7 4 cd74 antigen (invariant polpypeptide of major 1.0 a 1.8 b

histocompatibility class ii antrgen-associated)

---- Ig active lambda2-like chain 1.0 a 1.7 b
Hla-drnb rtl class ii, locus dmb 1.0 a 1.7 b
---- NP defensin 3 alpha precru’sor 1.0 a 1.7 b
RTl-Da rtl class ii, locus da 1.0 a 1.6 b
RTl-Ba rtl class ii, locus ba 1.0 ‘ 1.5 b
RTl-Bb rtl class ii, locus bb 1.0 a 1.5 b
C012 chemokine (c-c motif) ligand 2 1.0 a 1.4 b
Mif macrophage migration inhibitory factor 1.0 a 1.3 b
Psmb9 groteosome (prosome, macropain) subumt, beta type 1.0 a 1.3 b
Mxl myxovirus (influenza virus) resistance 1.0 a 1.3 b
RTl-M3 n1 class ib, locus m3 1.0 a 1.3 b
1115 interleukin 15 1.0 b 0.6 “

 

 

104

 

TABLE 3. Select genes differentially expressed by carcinogen (AOM) treatment
in the distal colonic epithelium of male F344 rats. (continued) 1

Gene symbol

 

Common

III. Intracellular protein metabolism

Saline- AOM-
Injected Injected

Rps7 ribosomal protein $7 1.0 a 1.4 b
RpslS ribosomal protein 815 1.0 a 1.4 b
Hspel heat shock 10 kda protein 1 1.0 a 1.3 b
Rpl37 ribosomal protein 137 1.0 a 1.3 b
Rpl4 ribosomal protein 14 1.0 a 1.3 b
Psmb4 proteasome (prosome, macropain) subunit, beta type 1.0 a 1.3 b
Rps4x ribosomal protein s4, x-linked 1.0 a 1.3 b
Rpsl7 ribosomal protein 817 1.0 a 1.3 b
Rpl36a large subunit ribosomal protein 136a 1.0 ' 1.3 b
Rps9 ribosomal protein $9 1.0 ‘ 1.3 b
Pcsk3 proprotein convertase subtilisin/kexin type3 1.0 ‘ 1.3 b
Pam peptidylglycine alpha-amidating monooxygenase 1.0 b 0.6 “
Rps24 ribosomal protein 524 1.0 b 0.6 ‘

 

 

 

1 Data expressed as mean-fold change differences standardized to the SHAM (saline-

injected) animals (n=12/group). Superscripts denote significant main effects for

carcinogen treatment (P < 0.05 2.

105

Genes differentially apressed by dietary treatment.

Among known transcripts differentially affected by dietary treatment (n=202),
compared to the AIN diet, feeding rats black beans (BB) significantly affected 145 genes
(90 up-regulated, 55 down-regulated) and soy flour (SF) affected 115 genes (87 up-
regulated, 28 down-regulated). Fifty genes were significantly co-induced and 22 co-
repressed by BB and SF, representing 36% of gene changes, although an additional 30%
showed the same direction of change. A majority of known transcripts affected by
dietary treatment (54%) fell into one of five categories including other (n=31, 15%),
enzymes (n=30, 15%), energy metabolism (n=24, 12%), channel, transporter, carrier
proteins (n=22, 11%), and cell cycle and apoptosis (n=21, 10%) (FIGURE 2).

A select listing of transcripts similarly affected in the colon of bean-fed compared
to casein-fed rats is presented in TABLE 4, and a full listing of the remaining transcripts
can be found in the APPENDICES as SUPPLEMENTARY TABLE 3. As shown in
TABLE 4, bean-based diets co-induced a number of genes involved in fatty acid
metabolism and gluconeogenesis (HMG CoA synthase 2, PEPCK, enoyl CoA hydratase
l), electron transport (CYPp450 4b1&2d1, diaphorase 1), detoxification (gluthionine s
transferase, UDP glycotransferases, perixoredoxin) and solute/ion transport (aquaporin 8,
sodium channel, solute carriers). Among genes co-repressed included some cell cycle
associated proteins (cyclin B1, CDC2), fatty acid desaturation enzymes (stearoyl Co-A
desaturase 1&2), extracellular matrix proteins (collagen 10L, fibronectin 1), innate
immune response/inflammatory proteins (MIF, sPLA2, NP defensin 3a, toll-like receptor

4, cdldl antigen), and proteins involved in nucleic acid binding/transcription regulation

(high mobility group box 2, early grth response 1 & 2).

106

 

 

TABLE 4. Genes similarly affected by bean-feeding (BB and SF) in the distal
colonic epithelium of male F344 rats. 1

 

 

Gene

symbol Gene Title AIN 33 SF
1. Cell cycle_, cell growth and maintenancg, apoptosis
Ceacaml Icnacrflz‘ier:Ziegnlbryonic antigen-related cell adhesion 1.0 a 2.3 b 1.9 b
Rbl retinoblastoma 1 1.0 a 2.2 b 2.2 b
Sgk serum/glucocorticoid regulated kinase 1.0 a 1.9 b 1.6 b
Gad d4 5a 5:31:11 arrest and dna—damage-inducible 45 1.0 , 1.5 b 1.5 b
Txnip upregulated by 1,25-dihydroxyvitamin d-3 1.0 a 1.4 b 1.3 b
Csnkld casein kinase 1, delta 1.0 a 1.3 b 1.2 b
Bax bch-associated x protein 1.0 a 1.3 b 1.4 b
Egln3 egl nine homolog 3 (c. elegans) 1.0 b 0.7 a 0.8 “b
Wfdcl ' wap four-disulfide core domain 1 1.0 b 0.7 a 0.5 1'
Ccnbl cyclin bl 1.0 b 0.7 a 0.8 8"
Cd02a cell division cycle 2 homolog a (s. pombe) 1.0 b 0.6 ‘ 0.8 b
Top2a topoisomerase (DNA) 2 alpha 1.0 b 0.6 ‘ 0.8 b
11. Channel, transporters, & carriers
quS aquaporin 8 1.0 a 2.7 b 2.9 b
Scnnl g sodium channel, nonvoltage-gated 1 gamma 1.0 “ 2.2 b 2.0 b
--- apolipoprotein a-i 1.0 a 1.6 “b 2.4 b
--- k-cl cotransporter kcc4 1.0 a 1.6 b 1.4 b
Tfrc transferrin receptor 1.0 a 1.5 b 1.2 3"
SlcSal solute carrier family 5, member 1 1.0 a 1.5 b 1.3 3"
Slcl6a1 solute carrier family 16, member 1 1.0 a 1.5 b 1.5 b
Atplal atpase, na+k+ transporting, alpha 1 1.0 a 1.4 b 1.3 b
Lgals9 lectin, galactose binding, soluble 9 1.0 a 1.3 b 1.3 b
Kcnkl putative potassium channel twik 1.0 a 1.3 b 1.3 b
Slcl6a6 figgsgg figzelr‘zmmmmbmync “id 1.0 b 0.6 a 0.8 b
Fabp5 fatty acid binding protein 5, epidermal 1.0 b 0.5 a 0.6 “
--- tocopherol (alpha) transfer protein 1.0 b 0.5 ‘ 0.7 a

107

 

TABLE 4. Genes similarly affected by bean-feeding (BB and Soy) in the distal
colonic epithelium of male F344 rats. (continued) 1
Gene
symbol Gene Title AIN BB SF
111. Electron transpor_t, oxidoreductase, detoxification
Cyp4bl cytochrome p450, subfamily 4b, polypeptide 1 1.0 a 3.6 b 2.2 c

--- udp glycosyltransferase 1 family, polypeptide a1 1.0 a 1.7 b 2.2 b

 

Gsth glutathione s-transferase, mu 5 1.0 a 1.5 b 1.5 b
Gstrnl glutathione s-transferase, mu 1 1.0 a 1.4 b 1.3 3"
Prdx6 peroxiredoxin 6 1.0 a 1.4 “b 1.5 b
Cyp2d10 cytochrome p450 2d1 1.0 a 1.4 b 1.4 b
Sultlal :éprerpjferase family 1a, phenol-preferring, 1.0 a 1.4 o 1.2 ,b
For p450 (cytochrome) oxidoreductase 1.0 a 1.4 b 1.4 b
Cbe cytochrome b5 1.0 a 1.4 b 1.3 b
Etfdh electron-transferring-flavoprotein dehydrogenase 1.0 ° 1.4 b 1.2 3"
Tst thiosulfate sulfurtransferase 1.0 ‘ 1.3 b 1.3 b
Dial diaphorase 1 1.0 a 1.2 ab 1.3 b
Cbrl carbonyl reductase 1 1.0 a 1.2 ab 1.5 b

--- udp glycosyltransferase 1 family, polypeptide a7 1.0 a 1.2 b 1.3 b
--- udp glycosyltransferase 1 family, polypeptide a6 1.0 a 1.2 a 1.5 b

IV. Energy metabolism
3-hydroxy-3-methylglutaryl-coenzyme a

ngcsZ synthase 2 1.0 a 2.2 b 2.1 b
Aldob aldolase b 1.0 a 2.1 b 1.8 b
--- phosphoenolpyruvate carboxykinase 1 1.0 a 2.0 b 1.8 b
Echl enoyl coenzyme a hydratase 1 1.0 a 1.6 b 1.6 b
Glul glutarnine synthetase 1 1.0 a 1.4 ° 1.2 1’

hydroxyacyl-coenzyme a dehydrogenase/3-
Hadhb ketoacyl-coenzyme a thiolase/enoyl-coenzyme a 1.0 a 1.3 b 1.3 b
hydratase (trifunctional protein), beta subunit

Hsd17b2 l7-beta hydroxysteroid dehydrogenase type 2 1.0 a 1.2 a” 1.4 b
Sqle squalene epoxidase 1.0 a 1.2 “b 1.3 b
Mtel mitochondrial acyl-coa thioesterase 1 1.0 ’ 1.2 ab 1.8 b

 

108

 

TABLE 4. Genes similarly affected by bean-feeding (BB and Soy) in the distal
colonic epithelium of male F344 rats. (continued) '

 

 

 

Gene

symbol Gene Title AIN BB SF

IV. Energy metabolism
hydroxyacyl-coenzyme a dehydrogenase/3-
Hadha ketoacyl-coenzyme a hiolase/enoyl-coenzyme a 1.0 a 1.2 b 1.3 b
hydratase (trifunctional protein), alpha subunit

--- phosphofi'uctokinase, platelet 1.0 b 0.7 a 0.8 a
Gde glycerol-3-phosphate dehydrogenase 2 1.0 b 0.7 a 0.8 ab
Acsl4 fatty acid coenzyme a ligase, long chain 4 1.0 b 0.6 a 0.8 “b
peptide YY 1.0 b 0.6 a 0.7 “
--- glucagon 1.0 b 0.6 a 0.8 3"
Scd2 stearoyl-coenzyme a desaturase 2 1.0 b 0.5 a 0.7 3"
Scdl stearoyl-coenzyme a desaturase l 1.0 b 0.5 a 0.7 ab
V. Extracellular matrix, cell adhesion, cfloskeleton
--- cd36 antigen-like 2 1.0 a 1.4 b 1.2 3"
Sdcl syndecan 1 1.0 a 1.3 b 1.3 b
Fnl fibronectin 1 1.0 b 0.7 a 0.6 ‘
Tubb5 tubulin, beta 5 1.0 b 0.6 a 0.9 b
Strnnl stathmin 1 1.0 b 0.6 a 0.9 3"
Collal collagen, type 1, alpha 1 1.0 b 0.5 a 0.5 a
VI. Immune, defense, inflammation, stress
Hspala heat shock 70kd protein 1a 1.0 a 1.9 b 1.4 8"
RTl-Aw2 rtl class ib, locus aw2 1.0 a 1.5 b 1.4 b
Tff3 trefoil factor 3 1.0 a 1.5 b 1.2 ab
RTl-Aw2 mhc class i rt1.o type 149 processed pseudogene 1.0 a 1.3 b 1.2 b
--- protease (prosome, macropain) 28 subunit, beta 1.0 a 1.3 b 1.2 ab
Tlr4 toll-like receptor 4 1.0 b 0.8 a 0.7 “
Mif macrophage migration inhibitory factor 1.0 b 0.7 ° 0.9 3"
Dmbtl deleted in malignant brain tumors 1 1.0 b 0.6 a 0.7 “
Pla2g2a phospholipase a2, group iia 1.0 b 0.5 a 0.4 “
--- NP defensin 3 alpha precursor 1.0 b 0.5 a 0.7 “

 

109

 

TABLE 4. Genes similarly affected by bean-feeding (BB and Soy) in the distal
colonic epithelium of male F344 rats. (continued) '

 

 

Gene

symbol Gene Title AIN 33 SF
VII. Nucleic acid binding, transcription regulation
Nr1d2 nuclear receptor subfamily 1, group (1, member 2 1.0 a 2.2 b 1.3 “
Pem placentae and embryos oncofetal gene 1.0 a 1.6 b 1.3 “b
Nfib nuclear factor i/b 1.0 a 1.5 b 1.4 b
Vdr vitamin (I receptor 1.0 a 1.4 “b 1.6 b
del synaptotagmin 4 1.0 b 0.8 “b 0.6 “
Egr2 early grth response 2 1.0 b 0.8 a 0.6 ‘
Gata6 gata binding protein 6 1.0 b 0.7 a 0.9 3"
Nr4a2 nuclear receptor subfamily 4, group a, member 2 1.0 b 0.7 a 0.7 “
ngb2 high mobility group box 2 1.0 b 0.7 " 0.9 b
Egrl early growth response 1 1.0 b 0.5 a 0.7 3
VIII. Signal transduction
Guca2a guanylate cyclase activator 2a 1.0 a 1.5 b 1.4 3"
Csrp2 cysteine-rich protein 2 1.0 a 1.5 b 1.5 b
Sstr2 somatostatin receptor 2 1.0 a 1.5 b 1.7 b
Mapk14 mitogen activated protein kinase 14 1.0 a 1.3 b 1.4 b
Gucy20 guanylate cyclase 20 1.0 a 1.3 b 1.4 b
P2ry2 purinergic receptor p2y, g-protein coupled 2 1.0 b 0.8 “b 0.6 “
P2ry6 pyrimidinergic receptor p2y, g-protein coupled, 6 1.0 b 0.7 ° 0.7 ‘
Pldl phospholipase d1 1.0 b 0.7 a 0.9 b
Ptpro protein tyrosine phosphatase, receptor type c 1.0 b 0.6 a 0.7 ‘
--- frizzled homolog 1 (drosophila) 1.0 b 0.6 ‘ 0.7 8
IX. Engines, other
Plat plasminogen activator, tissue 1.0 a 3.0 b 2.4 ab
Abat 4-aminobutyrate aminotransferase 1.0 a 3.0 ° 1.7 b
Ca4 carbonic anhydrase 4 1.0 a 1.7 b 1.7 b

protein phosphatase 1b, magnesium dependent, , b 3,,

--- beta isoforrn 1 '0 1'6 1 '4
Ptpnl protein tyrosine phosphatase, non-receptor type 1 1.0 “ F1.6 b 1.3 3"

 

110

 

 

TABLE 4. Genes similarly affected by bean-feeding (BB and Soy) in the distal
colonic epithelium of male F344 rats. (continued) '

 

 

 

 

Gene

symbol Gene Title AIN 33 31?
TX. Enzymes, other -
Oasl 25 oligoadenylate synthetase 1.0 a 1.5 b 1.5 b
Dpp4 dipeptidylpeptidase 4 1.0 a 1.4 b 1.3 b
Cyp27a1 3:33:32: p450, family 27, subfamily a, 1.0 a 1.4 b 1.5 b
Ache acetylcholinesterase 1.0 a 1.4 b 1.5 b
Adk adenosine kinase 1.0 a 1.3 b 1.5 b
Pts 6-pyruvoyl-tetrahydropterin synthase 1.0 a 1.3 “b 1.5 b
Gcnt3 beta-1,6-n-acetylglucosaminyltransferase 1.0 b 0.7 a 0.7 a
Ctse cathepsin e 1.0 b 0.7 a 0.7 a
X. Other
Map17 membrane-associated protein 17 1.0 a 2.7 b 1.4 “
Zg16 zymogen granule protein 16 1.0 a 2.2 b 1.9 1’
Spinkl atpase inhibitor 1.0 a 2.1 b 1.4 “
--- germinal histone h4 gene 1.0 a 2.0 b 1.4 3"
Gchfr gtp cyclohydrolase i feedback regulatory protein 1.0 a 1.9 b 1.8 b
calbindin 3 1.0 a 1.6 b 1.2 a
Mucl mucin 1 1.0 a 1.6 ° 1.3 b
Ensa endosulfine alpha 1.0 ° 1.5 b 1.4 3"
F3 coagulation factor 3 1.0 b 0.7 a 0.5 a
annexin 5 1.0 c 0.5 a 0.7 b
“Mb“: ‘ _ 1 0. . . . . 6“

Data expressed as mean-fold change differences standardized to the AIN group
(n=8/Eo up). Superscripts denotel iguficant main effects _for diet (P < 0.05).

 

 

 

111

Genes affected by dietary treatment and carcinogen injection.

Nine genes were found to be influenced both by diet and carcinogen and are
presented in TABLE 5. Transcripts for collagen 10:1, fibronectin 1, NP defensin 3a, and
sPLA2 were induced by carcinogen treatment, but repressed in rats fed either BB or SF.
The expression for these genes was highest in the AIN(AOM-inj ected) group, and tended
to parallel tumor incidence data. AOM-injected animals had a lower abundance of genes
encoding somatostatin 2 receptor, carbonyl reductase, and amyloid beta (a4) precursor
protein-binding, family b, member 3. The two former were generally higher in both BB
and SF groups whereas the latter was elevated in the SF group only. Seven other
transcripts occurred in both lists, however annotations were different, and when averaged,
did not reach statistical significance for both diet and injection.

RT -PCR gene changes.

The abundance of five selected genes (CDC2, cyclin B1, TOP2A, GADD450t,
and sPLA2) was confirmed by RT-PCR. These genes were chosen due to the known
effects of cell proliferation and inflammation on tumorigenesis. As shown in FIGURE
2, the mRNA for CDC2, cyclin B1, and TOP2A were all significantly lower, whereas
GADD450t was higher in the colon of BB and SF-fed rats compared to controls (AIN).
The relative fold changes in sPLA2 are presented in FIGURE 3. In accordance with
microarray data, there were significant main effects for both diet (P < 0.009) and
carcinogen (P < 0.006) treatment. Generally, sPLA2 expression was lowest in bean-fed

rats, but increased in all diets with carcinogen injections (fold-increases of 2.6,

112

 

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113

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114

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Relative fold-change

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

AIN BB SOY
FIGURE 3. Relative fold-changes in sPLA2 mRNA abundance detected
with RT-PCR. Results were normalized to the housekeeping gene B-Actin

and are presented as mean fold-changes (LSM i SEM) relative to the

AIN(saline-injected) group. There were significant main effects for both
diet (P < 0.009) and injection type (P < 0.006) on sPLA2 abundance.

Superscripts denote significant differences between groups (P < 0.05).

115

3.1, and 3.1 for AIN, BB, and SF fed rats, respectively). Rats injected with AOM and fed
the control (AIN) diet had the highest (2.6) overall expression level whereas mRNA
expression in the BB(AOM-injected) (1.2) and SF (AOM-injected) (1.0) groups increased

to values that were not different from the AIN(saline-injected) group (1.0).

_E_. DISCUSSION.

The focus of the current research was to ascertain potential cellular and molecular
events underlying suppression of tumorigenesis by beans using a highly relevant animal
model of colon cancer. We profiled global changes in gene expression affected by
AOM-treatment in non-neoplastic colon mucosa to determine early events permissive for
tumor formation and whether these changes could be attenuated by dietary treatment.
Although not a primary focus of this study, tumor incidence was also assessed. As
previously demonstrated, both BB (12) and SF-fed (8-11) rats developed significantly
fewer tumors overall, confirming that these diets inhibit experimental colon
carcinogenesis. In the study by Hangen and Bennink (12), however, black and navy bean
fed-rats ate less, and as a result had significantly lower body weights at termination of the
study. Because of the inverse association between energy restriction and tumorigenesis,
the tryptophan and methionine content in the black bean diets was adjusted to raise the
amino acid score comparable to that of the AIN and SF diets. As a result, no significant
differences in final weight gain were detected, indicating that black beans inhibit
tumorigenesis by a mechanism other than energy restriction.

Despite extensive use of the AOM model to study colon carcinogenesis, only a

few studies have examined the molecular events associated with colon cancer

116

development. Davidson et al. (37) studied colon tissue 12 hours and 18 weeks after
AOM injection, and suggested that AOM treatment caused changes in genes involved in
apoptosis, although actual data were not presented. Other studies have examined
molecular changes in microscopic lesions in the colon of carcinogen treated animals. For
example, aberrant crypt foci (ACF) are lesions containing crypts that are larger in size,
have altered lumenal openings, and exhibit thickened epithelia (38). ACF have been
reported to occur in a dose-dependent manner following AOM-administration (3 8) and
contain a high frequency of mutations in the proto-oncogene k-ras (29, 32). Activating
mutations in B-catenin have also been reported in more dysplastic ACF, but nuclear
accumulation of the protein is not frequently observed (39). Although ACF are
considered to be putative precursors to adenomas, many ACF disappear and never
develop into adenomas (40-41). On the other hand, Yarnada and collegues (39, 42-45)
recently described the presence of B-catenin accumulated crypts (BCAC) in en face
preparations of rat colons following AOM-administration. BCAC are distinct from ACF,
contain mutations in and nuclear accumulation of B-catenin, and exhibit disruptions in
cellular morphology, dysplasia, and increased proliferative capacity (39, 42-45).
Mutations affecting degradation of B-catenin or inactivation of the adenomatous
polyposis coli (APC) gene are accepted to be initiating events in human (46-47) and rat
colon tumorigenesis (31-32), respectively, thus BCAC may represent true premalignant
lesions.

In this study, the global effects of AOM at 31 weeks post-treatment were
associated with a higher expression of genes involved in innate defense and immunity.

For example, the anti-microbial genes lysozyme and neutrophil (NP) defensin 30L were

117

'I

approximately 2-fold higher than in saline injected controls. Lysozyme expression is
characteristic of Paneth cell lineage, which are rarely found in the colon, but have been
detected in dysplastic ACF (35), BCAC (42-44) and colon tumors (35). The presence of
Paneth cells has been suggested to be a consequence of altered differentiation
programming, possibly through aberrant activation of the Wnt/B-catenin pathway (43,
48). Several major histocompability MHC II-associated antigens were also induced in
the colon of AOM—injected rats. Epithelial cells, activated dendritic cells, and/or
macrophages underlying the intestinal cell layer can present MHC II antigens (49-52). A
higher abundance of these transcripts implies enhanced immune responsiveness to
lumenal antigens and suggests that AOM treatment alters mucosal barrier function. Soler
(53) noted defects in tight-junction permeability in normal mucosa from carcinogen-
treated animals, and the surface epithelial cells of ACF have also been reported to be
deficient in mature goblet cells, have altered mucin composition, and contain irregular
microvilli (54). A defective mucosal barrier that allows more antigenic materials to pass
through the underlying lamina propria would be expected to result in low-grade
inflammation and chronic inflammation has been implicated in the etiology of colon
cancer (SS-56). Although none of the well-known cell cycle-associated genes were
altered by AOM-treatment, we detected moderate induction of ribosomal proteins,
especially components of the 40S and 60S subunits. RNA and protein synthesis have
been reported to decrease as cells terminally differentiate (57), and enhanced presence of
ribosomal proteins has been noted in the colon of rat strains susceptible to PhIP-induced

colon cancer (58) and in animals during aging (59).

118

The profile of genes altered by dietary treatment highlight differences in colon
cell physiology with particular implications to carcinogenesis. For example, the most
highly induced classes of genes affected by bean-diets included those involved in water
channel and ion transport, oxidative electron transport, and fatty acid metabolism. These
changes are consistent with the physiological effects of fermentable fibers in bean diets
on colon epithelial function. Bacterial fermentation of dietary fiber and resistant starch
produces the short-chain fatty acids (SCFA), acetate, propionate, and butyrate (60-61).
SCFAs are known to be trophic to the colonic epithelium, enhance water and sodium
absorption, increase mucosal blood flow, and modulate enterohormone release (reviewed
in 62-64). Butyrate in particular has been demonstrated to inhibit cancer cell growth in
vitro (65-66), and it has been suggested that butyrate metabolism, through mitochondrial
B-oxidation, is important for induction of apoptosis in vivo (67). Downregulation of
mitochondrial genes have been demonstrated in inflammatory conditions (36, 68),
although it is not clear if this is a cause or effect relationship.

Alterations in crypt cellular kinetics associated with an expansion of the
proliferative compartment towards the lumen has been described in non-neoplastic colon
tissue fi'om humans at risk for colon cancer development and in animals treated with
carcinogens (69). In this study, several transcripts associated with proliferation and
apoptosis were found to be regulated in a diet-dependent manner. An interesting finding
was a 2-fold higher expression of CEACAM] in rats fed either BB or SF. CEACAMl is
a cell-surface glycoprotein expressed in the differentiated cell compartment of colonic
crypts that has been proposed to induce apoptotic signals in response to ankinois (70-71).

Loss of CEACAMI expression occurs with a similar frequency in hyperplastic polyps

119

and human colon cancers, and appears to precede defects in the APC pathway (71).
Somatostatin receptor 2, which mediates anti-proliferative responses to the endocrine
hormone somatostatin (72-73), was also more abundant in bean-fed animals and tended
to decrease following AOM treatment. Whey and soy protein isolate have been reported
to enhance both colonic mRNA and serum protein levels of somatostatin, suggesting
neuroendocrine involvement in epithelial kinetics (74). Additionally, we detected lower
mRNA abundance for the mitotic genes cyclin B1, CDC2, and TOP2A and higher
GADD450L in bean-fed rats, indicative of a GZ/M cell cycle arrest (75). TOP2A is
involved in a variety of processes including DNA replication, chromosome segregation,
and maintenance of chromosome structure (76). Protein expression has been detected
primarily in the lower crypt compartment of human colons and levels increase during
tumorigenesis (77). It is interesting that expression of these genes can be regulated in a
p53-dependent manner (reviewed in 75), suggestive of enhanced capability for DNA
repair and/or apoptotic responses in bean-fed animals. The anatomical distribution of
TOP2A and CEACAMI along the crypt-lumen axis concurrent with dietary modulation
of other cell-cycle associated genes implies a general effect of bean diets on maintaining
crypt cell homeostasis, potentially through enhancing cellular differentiation.

Of particular importance in this study with respect to dietary modulation of
tumorigenesis were genes changes that paralleled tumor incidence data. Specifically,
transcripts for sPLA2, NP defensin 30c, collagen la], and fibronectin l were induced by
AOM treatment in all diets, but less so in bean-fed animals. sPLA2 is induced in a
variety of inflammatory and neoplastic conditions in the colon and other tissues (78-85).

The protein exhibits anti-microbial activity (86), releases arachidonic acid from lipid

120

membranes (87-88), and has been demonstrated to activate neutrophils through catalytic
mechanisms (arachidonic acid liberating) and independent of catalytic activity in vitro
(89). Ikegami et al. (81) reported that in rats, colonic sPLA2 mRNA, but not cPLA2 or
COX-2, increases acutely following carcinogen administration and is associated with
higher levels of PGE2 and 6keto PGF 1a. Production of prostaglandins would likely
contribute to colon carcinogenesis through known effects on epithelial cell proliferation,
apoptosis, and angiogenesis (90-93). Defensins are small cationic peptides, divided into
or and [3 groups depending on the position of the inter-disulfide bonds between cysteine
residues (94), and exhibit a range of antimicrobial acivity in vitro (95). NP defensin 3a
was originally identified in neutrophil granules (96), however it has been detected in
epithelial cells during severe inflammation (97) and in colon cancers (35). Similar to
sPLA2, NP defensin 30. was induced by AOM injections in all diets, but levels were
highest in AlN(casein)-fed animals. These results, in addition to a lower expression of
toll-like receptor 4, a component of innate defense that initiates inflammation in response
to bacterial lipopolysaccharide (98), implies that bean-feeding protects against AOM-
induced mucosal barrier dysfunction.

The extracellular matrix (ECM) proteins fibronectin and collagen are involved in
a variety of processes including proliferation, cell adhesion, and chemotaxis. Actively
proliferating epithelial cells at the crypt base synthesize more fibronectin and levels
decrease during differentiation (99). In addition, fibronectin has been identified as a
downstream target of the Wnt/B-catenin pathway (100-101). Collagen locl expression is
upregulated in colon cancer (CHAPTER III) and in other hyperproliferative disorders

(102), but normal colonic expression has not been reported. Dietary modulation of these

121

genes may represent a lower presence of BCAC and/or dysplastic cypts in bean-fed
animals or a more general effect of diet on enhancing cellular differentiation.

In summary, this study provided insight into early molecular events associated
with AOM-induced colon cancer as well as in chemoprevention by bean feeding. One of
the major findings with respect to AOM-treatment was a higher expression of immune-
related antigens, suggestive that dysfimctions in mucosa integrity are permissive for
colon carcinogenesis. Bean-feeding induced changes in gene profiles consistent with
modulation of crypt cytokinetics and energy metabolism. Specific molecular targets of
beans that appear to corroborate reduced tumorigenesis include suppression of the genes
sPLA2 and NP defensin 3a, as well as alterations in ECM components, which may affect
migration and/or differentiation programming. We speculate that these gene changes
coincide with the ability of bean diets to protect against mucosal barrier dysfunction,
thereby suppressing microbial-induced inflammation. Future studies examining this

possibility in more detail are warranted.

122

L

10.

11.

12.

LITERATURE CITED.

American Cancer Society. (2005) Cancer Facts & Figures 2005.
http://www.cancer.or,g1downloads/STT/CAFFZOO5f4PWSecured.pdf.

American Institute of Cancer Research/World Cancer Research Fund
(AICR/WCRF). (1997) Colon, rectum. In Food, Nutrition and the Prevention of
Cancer: A Global Perspective, BANT A Book Group, Menasha, WI, pp. 216-51.

Potter JD. (1999) Colorectal cancer: Molecules and populations. Journal of the
National Cancer Institute. 91 (1 1): 916-932.

Steinmetz KA and Potter JD. (1993) Food-group consumption and colon cancer in
the Adelaide Case-Control Study. 1. Vegetables and fruit.
Int J Cancer. 53(5): 711-9.

Singh P N and Fraser GE. (1998) Dietary risk factors for colon cancer in a low-risk
population. American Journal of Epidemiology. 148: 761-774

Fraser GE. (1999) Associations between diet and cancer, ischemic heart disease,
and all—cause mortality in non-Hispanic white California Seventh-day Adventists.
American Journal of Clinical Nutrition. 70: 532S-538S

Correa P. (1981) Epidemiological correlations between diet and cancer frequency.
Cancer Research. 41: 3685-3689

Bennink MR and Om AS. (1998) Inhibition of colon cancer (CC) by soy
phytochemicals but not by soy protein. F aseb Journal. 12(5): 3808.

Bennink MR, Om AS and Miyagi Y. (1999) Inhibition of colon cancer (CC) by soy
flour but not by genistin or a mixture of isoflavones. Faseb Journal. 13(4): A50-
A50.

Bennink MR, Om AS and Miyagi Y. (2000) Inhibition of colon cancer (CC) by
wheat bran, soy flour, and flax. Faseb Journal. 14(4): A217-A217.

Rondini EA and Bennink MR. (2002) Defatted soy flour, but not soy concentrate,

reduces azoxymethane-induced colon carcinogenesis. Journal of Nutrition. 132(3):
589S-589S.

Hangen LA and Bennink MR. (2003) Consumption of black beans and navy beans

(Phaseolus vulgaris) reduced axozymethane-induced colon cancer in rats. Nutrition
and Cancer. 44: 60-6

123

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Hughes JS, Ganthavom C, and Wilson-Sanders S. (1997) Dry beans inhibit
azoxymethane-induced colon carcinogenesis in F344 rats. Journal of Nutrition.
127: 2328-2333.

Hakkak R, Korourian S, Ronis MJJ et al. (2001) Soy protein isolate consumption
protects against azoxymethane-induced colon tumors in male rats. Cancer Letters.
166(1): 27-32.

Raicht RF, Cohen BI, Fazzini EP et al. (1980) Protective effect of plant sterols

against chemically induced colon tumors in rats.
Cancer Res. 40(2): 403-5.

Weed HG, McGandy RB and Kennedy AR. (1985) Protection against
Dimethylhydrazine-Induced Adenomatous Tmnors of the Mouse Colon by the
Dietary Addition of an Extract of Soybeans Containing the Bowman-Birk Protease
Inhibitor. Carcinogenesis. 6(8): 1239-1241.

Shamsuddin AM, Elsayed AM and Ullah A. (1988) Suppression of Large Intestinal
Cancer in F344 Rats by Inositol Hexaphosphate. Carcinogenesis. 9(4): 577-580.

Shamsuddin AM and Ullah A. (1989) Inositol Hexaphosphate Inhibits Large
Intestinal Cancer in F344 Rats 5 Months after Induction by Azoxymethane.
Carcinogenesis. 10(3): 625-626.

Ullah A and Shamsuddin AM. (1990) Dose-Dependent Inhibition of Large
Intestinal Cancer by Inositol Hexaphosphate in F344 Rats. Carcinogenesis. 11(12):
2219-2222.

Billings PC, Newbeme PM and Kennedy AR. (1990) Protease Inhibitor
Suppression of Colon and Anal Gland Carcinogenesis Induced by
Dimethylhydrazine. Carcinogenesis. 1 1(7): 1083-1086.

Koratkar R and Rao AV. (1997) Effect of soya bean saponins on azoxymethane-
induced preneoplastic lesions in the colon of mice. Nutrition and Cancer-an
International Journal. 27(2): 206-209.

Fotsis T, Pepper MS, Aktas E et al. (1997) Flavonoids, dietary-derived inhibitors of
cell proliferation and in vitro angiogenesis. Cancer Res. 57: 2916-2921.

Wenzel H, Kuntz S, Brendel MD and Daniel H. (2000) Dietary flavone is a potent
apoptosis inducer in human colon carcinoma cells. Cancer Research. 60(14): 3823-
3831.

Nguyen DT, Garcia J and Cadenas E. (2001) Genistein causes cell cycle arrest and
apoptosis via inhibition of p53-independent mechanisms in T47D breast cancer
cells. Free Radical Biology and Medicine. 31: ($105-$105).

124

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Sandra F, Matsuda M, Yoshida H and Hirata M. (2002) Inositol hexakisphosphate
blocks tumor cell growth by activating apoptotic machinery as well as by inhibiting

the Akt/NF kappa B-mediated cell survival pathway. Carcinogenesis. 23(12):
2031-2041.

Kennedy AR, Billings PC, Wan XS and Newbeme PM. (2002) Effects of Bowman-
Birk inhibitor on rat colon carcinogenesis. Nutrition and Cancer-an International
Journal. 43(2): 174-186.

Yu ZL, Li W] and Liu FY. (2004) Inhibition of proliferation and induction of
apoptosis by genistein in colon cancer HT-29 cells. Cancer Letters. 215(2): 159-
166.

Jacoby RF, Llor X, Teng BB et al. (1991) Mutations in the K-Ras Oncogene
Induced by 1,2-Dimethylhydrazine in Preneoplastic and Neoplastic Rat Colonic
Mucosa. Journal of Clinical Investigation. 87(2): 624-630.

Vivona AA, Shpitz B, Medline A et al. (1993) K-Ras Mutations in Aberrant Crypt
Foci, Adenomas and Adenocarcinomas During Azoxymethane-Induced Colon
Carcinogenesis. Carcinogenesis. 14(9): 1777-1781.

Pories SE, Ramchurren N, Summerhayes I and Steele G. (1993) Animal models for
colon carcinogenesis. Archives of Surgery. 128: 647-53.

Takahashi M, Fukuda K, Sugimura T and Wakabayashi K. (1998) Beta-catenin is
frequently mutated and demonstrates altered cellular location in azoxymethane-
induced rat colon tumors. Cancer Research. 58(1): 42-46. ACF

Takahashi M and Wakabayashi K. (2004) Gene mutations and altered gene
expression in azoxymethane-induced colon carcinogenesis in rodents. Cancer
Science. 95(6): 475-480.

Liu T, Mokuolo A0, Rao CV et al. (1995) Regional chemoprevention of
carcinogen-induced tumors in rat colon. Gastroenterology. 109: 1167-72.

Reeves PG, Nielsen FH and Fahey GC. (1993) Ain—93 Purified Diets for
Laboratory Rodents - Final Report of the American Institute of Nutrition Ad Hoc
Writing Committee on the Reforrnulation of the Ain—76a Rodent Diet. Journal of
Nutrition. 123(11): 1939-1951.

Fujiwara K, Ochiai M, Ohta T et al. (2004) Global gene expression analysis of rat
colon cancers induced by a food-bome carcinogen, 2-amino-1-methyl-6-
phenylimidazo[4,5-b]pyridine. Carcinogenesis. 25(8): 1495- 1 505.

125

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

Lawrance IC, Fiocchi C and Chakravarti S. (2001) Ulcerative colitis and Crohn’s
disease: distinctive gene expression profiles and novel susceptibility candidate
genes. Human Molecular Genetics. 10(5): 445-56.

Davidson LA, Nguyen DV, Hokanson RM et al. (2004) Chemopreventive n-3
polyunsaturated fatty acids reprogram genetic signatures during colon cancer
initiation and progression in the rat. Cancer Research. 64(18): 6797-6804.

Bird RP. (2005) Role of aberrant crypt foci in understanding the pathogenesis of
colon cancer. Cancer Lett. 93: 55-71.

Yamada Y, Yoshimi N, Hirose Y et al. (2000) Frequent beta-catenin gene
mutations and accumulations of the protein in the putative preneoplastic lesions

lacking macroscopic aberrant crypt foci appearance, in rat colon carcinogenesis.
Cancer Research. 60(13): 3323-3327.

Hardman WE, Cameron IL, Heitrnan DW and Contreras E. (1991) Demonstration
of the Need for End-Point Validation of Putative Biomarkers - Failure of Aberrant
Crypt Foci to Predict Colon Cancer Incidence. Cancer Research. 51(23): 6388-
6392.

Davies MJ, Bowey EA, Adlercreutz H et al. (1999) Effects of soy or rye
supplementation of high-fat diets on colon tumour development in azoxymethane-
treated rats. Carcinogenesis. 20(6): 927-931.

Yamada Y, Yoshimi N, Hirose Y et al. (2001) Sequential analysis of
morphological and biological properties of beta-catenin-accumulated crypts,
provable premalignant lesions independent of aberrant crypt foci in rat colon
carcinogenesis. Cancer Research. 61(5): 1874-1878.

Yamada Y and Mori H. (2003) Pre-cancerous lesions for colorectal cancers in
rodents: a new concept. Carcinogenesis. 24(6): 1015-1019.

Hirose Y, Kuno T, Yamada Y et al. (2003) Azoxymethane-induced beta-catenin—
accumulated crypts in colonic mucosa of rodents as an intermediate biomarker for
colon carcinogenesis. Carcinogenesis. 24(1): 107-111.

Mori H, Yamada Y, Kuno T and Hirose Y. (2004) Aberrant crypt foci and beta-
catenin accumulated crypts; significance and roles for colorectal carcinogenesis.
Mutation Research-Reviews in Mutation Research; 566(3): 191-208.

Fearon ER and Vogelstein B. (1990) A Genetic Model for Colorectal
Tumorigenesis. Cell. 61(5): 759-767.

Kinzler KW and Vogelstein B. (1996) Lessons from hereditary colorectal cancer.
Cell. 87(2): 159-170.

126

 

 

48.

49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

Andreu P, Colnot S, Godard C et al. (2005) Crypt-restricted proliferation and
commitment to the Paneth cell lineage following Apc loss in the mouse intestine.
Development. 132(6): 1443-1451.

Nagashima R, Maeda K, Imai Y and Takahashi T. (1996) Lamina propria
macrophages in the human gastrointestinal mucosa: Their distribution,
immunohistological phenotype, and function. Journal of Histochemistry &
Cytochemistry. 44(7): 721-731 .

Marie 1, Holt PG, Perdue MH and Bienenstock J. (1996) Class II MHC antigen
(Ia)-bearing dendritic cells in the epithelium of the rat intestine. Journal of
Immunology. 156(4): 1408-1414.

German AJ, Bland PW, Hall EJ and Day MJ. (1998) Expression of major
histocompatibility complex class H antigens in the canine intestine. Veterinary
Immunology and Immunopathology. 61 (2-4): 171 -1 80.

Macdonald TT. (2003) The mucosal immune system. Parasite Immunology; 25(5):
235-246.

Soler AP, Miller RD, Laughlin KV et al. (1999) Increased tight junctional
permeability is associated with the development of colon cancer. Carcinogenesis.
20(8): 1425-1431.

Vaccina F, Scorcioni F, Pedroni M et al. (1998) Scanning electron microscopy of

aberrant crypt foci in human colorectal mucosa. Anticancer Research. 18(5A):
3451-3456.

O'Byme KJ and Dalgleish AG. (2001) Chronic immune activation and
inflammation as the cause of malignancy. Br J Cancer. 85(4): 473-83.

Huls G, Koomstra JJ and Kleibeuker JH. (2003) Non-steroidal anti-inflammatory
drugs and molecular carcinogenesis of colorectal carcinomas. Lancet. 362(9379):
230-232.

Deschner EE and Lipkin M. (1970) Study of Human Rectal Epithelial Cells in-
Vitro .3. Rna Protein and DNA Synthesis in Polyps and Adjacent Mucosa. Journal
of the National Cancer Institute. 44(1): 175-.

Fujiwara K, Ochiai M, Ubagai T et al. (2003) Differential gene expression profiles
in colon epithelium of two rat strains with distinct susceptibility to colon
carcinogenesis after exposure to PhIP in combination with dietary high fat. Cancer
Science. 94(8): 672-678.

127

59.

60.

61.

62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

Lee HM, Greeley GH and Englander EW. (2001) Age-associated changes in gene
expression patterns in the duodenum and colon of rats. Mechanisms of Ageing and
Development; 122(4): 355-371.

Cummings JH. (1981) Short chain fatty acids in the human colon. Gut. 22(9): 763-
79.

Cummings JH. and Macfarlane, GT. (1991) The control and consequences of
bacterial fermentation in the human colon. J. Appl. Bacter. 70: 443-59.

Cook SI and Sellin JH. (1998) Review article: short chain fatty acids in health and
disease. Alimentary Pharmacology & Therapeutics. 12(6): 499-507.

Blottiere HM, Champ M, Hoebler C, Michel C and Cherbut C. (1999) Production
and digestive effects of short chain fatty acids. Sciences Des Aliments. 19(3-4):
269-290.

Miller SJ. (2004) Cellular and physiological effects of short-chain fatty acids. Mini-
Reviews in Medicinal Chemistry. 4(8): 839-845.

Hague A, Manning AM, Huschtscha LI et al. (1993) Sodium butyrate induces
apoptosis in human colonic tumor cell lines in a p-53 independent pathway:
implications for the possible role of dietary fiber in the prevention of large bowel
cancer. Int. J. Cancer. 55: 498-505.

Heerdt BG, Houston MA, and Augenlicht LH. ( 1997) Short-chain fatty acid-
initiated cell cycle arrest and apoptosis of colonic epithelial cells is linked to
mitochondrial function. Cell Growth & Differentiation. 8: 523-32.

Augenlicht LH, Anthony GM, Church TL et al. (1999) Short-chain fatty acid
metabolism, apoptosis, and Apc-initiated tumorigenesis in the mouse
gastrointestinal mucosa. Cancer Research. 59(23): 6005-6009.

Birkenkamp-Demtroder K, Christensen LL, Olesen SH et. al. (2002) Gene
expression profiling in colorectal cancer. Cancer Research. 62: 4352-4363.

Lipkin M. (1974) Phase 1 and Phase 2 Proliferative Lesions of Colonic Epithelial-
Cells in Diseases Leading to Colonic Cancer. Cancer. 34(3): 878-888.

Shively JE. (2004) CEACAMl and hyperplastic polyps: new links in the chain of
events leading to colon cancer. Oncogene. 23(58): 9303-9305.

Nittka S, Gunther J, Ebisch C, Erbersdobler A and Neumaier M. (2004) The human

tumor suppressor CEACAMl modulates apoptosis and is implicated in early
colorectal tumorigenesis. Oncogene. 23(5 8): 9306-9313.

128

72.

73.

74.

75.

76.

77.

78.

79.

80.

81.

82.

83.

Buscail L, Esteve JP, Saintlaurent N et al. (1995) The Somatostatin Receptor
Subtype-Sstr2 and Subtype-SstrS Mediate the Inhibition of Cell-Proliferation.
Gastroenterology. 108(4): A955-A955.

Vemejoul F, F aure P, Benali N et al. (2002) Antitumor effect of in vivo
somatostatin receptor subtype 2 gene transfer in primary and metastatic pancreatic
cancer models. Cancer Research. 62(21): 6124-6131.

Xiao R, Badger TM and Simmen FA. (2004) Dietary exposure to soy or whey
proteins alters colonic global gene expression profiles during rat colon
tumorigenesis. Mol Cancer. 11: 4(1).

Taylor WR and Stark GR. (2001) Regulation of the G2/M transition by p53.
Oncogene. 20(15): 1803-1815.

Bakshi RP, Galande S and Muniyappa K. (2001) Functional and regulatory
characteristics of eukaryotic type 11 DNA topoisomerase. Critical Reviews in
Biochemistry and Molecular Biology. 36(1): 1-37.

Boman BM, Walters R, Fields JZ et al. (2004) Colonic crypt changes during
adenoma development in familial adenomatous polyposis - Immunohistochemical

evidence for expansion of the crypt base cell population. American Journal of
Patholog. 165(5): 1489-1498.

Nevalainen TJ. (1993) Serum Phospholipases A(2) in Inflammatory Diseases.
Clinical Chemistry. 39(12): 2453-2459.

Minami T, Tojo H, Shinomura Y et a1. (1994) Increased Group-Ii Phospholipase
a(2) in Colonic Mucosa of Patients with Crohns-Disease and Ulcerative-Colitis.
Gut. 35(11): 1593-1598.

Yarnashita S, Ogawa M, Sakamoto K et al. (1994) Elevation of Serum Group-Ii

Phospholipase a(2) Levels in Patients with Advanced Cancer. Clinica Chimica
Acta. 228(2): 91-99.

Ikegami T, Matsuzaki Y, Shoda J et al. (1998) The Chemopreventive role of
ursodeoxycholic acid in azoxymethane-treated rats: suppressive effects on

enhanced group II phospholipase A(2) expression in colonic tissue. Cancer Letters.
134(2): 129-139.

Haapamaki MM, Gronroos JM, Nunni H et al. (1997) Gene expression of group II
phospholipase A2 in intestine in ulcerative colitis. Gut. 40(1): 95-101.

Ogawa M. (1997) Group II phospholipase A(2) in neoplastic diseases.
Phospholipase A2. 24: ZOO-204.

129

84.

85.

86.

87.

88.

89.

90.

91.

92.

93.

94.

95.

96.

Haapamaki MM, Gronroos JM, Nurmi H et al. ( 1999) Phospholipase A(2) in serum
and colonic mucosa in ulcerative colitis. Scandinavian Journal of Clinical &
Laboratory Investigation. 59(4): 279-287.

Nevalainen TJ, Haapamaki MM and Gronroos JM. (2000) Roles of secretory
phospholipases A(2) in inflammatory diseases and trauma. Biochimica Et
Biophysica Acta-Molecular and Cell Biology of Lipids. 1488(1-2): 83-90.

Buckland AG and Wilton DC. (2000) The antibacterial properties of secreted

phospholipases A(2). Biochimica Et Biophysica Acta-Molecular and Cell Biology
of Lipids. 1488(1-2): 71-82.

Bingham CO and Austen KF. (1999) Phospholipase A(2) enzymes in eicosanoid
generation. Proceedings of the Association of American Physicians. 111(6): 516-
524.

Marshall LA and Morgan DW. (1998) Modulation of arachidonic acid metabolism:
Focus on phospholipase A(2)(s). Drug News & Perspectives. 11(2): 82-91.

Zallen G, Moore EE, Johnson JL et al. (1998) New mechanisms by which secretory
phospholipase A(2) stimulates neutrophils to provoke the release of cytotoxic
agents. Archives of Surgery. 133(11): 1229-1232.

Herschman HR. (1998) Recent progress in the cellular and molecular biology of
prostaglandin synthesis. Trends in Cardiovascular Medicine. 8(4): 145-150.

Ashktorab H, Dawkins FW, Mohamed R et al. (2005) Apoptosis induced by aspirin
and 5-fluorouracil in human colonic adenocarcinoma cells. Digestive Diseases and
Sciences. 50(6): 1025-1032.

Tarnawski AS and Jones MK. (2003) Inhibition of angiogenesis by NSAIDs:
molecular mechanisms and clinical implications. Journal of Molecular Medicine-
Jmm. 81(10): 627-636.

Williams CS, Mann M and DuBois RN. (1999) The role of cyclooxygenases in
inflammation, cancer, and development. Oncogene. 18(55): 7908-7916.

Cunliffe RN. (2003) alpha-Defensins in the gastrointestinal tract. Molecular
Immunology. 40(7): 463-467.

Ganz T and Lehrer RI. (1994) Defensins. Current Opinion in Immunology..6(4):
584-589.

Eisenhauer PB, Harwig SSL, Szklarek D et al. (1989) Purification and

antimicrobial properties of three defensins from rat neutrophils. Infect. Immun. 57:
2021-2027.

130

97.

98.

99.

100.

101.

102.

Cunliffe RN, Kamal M, Rose F et al. (2002) Expression of antimicrobial neutrophil
defensins in epithelial cells of active inflammatory bowel disease mucosa. Journal
of Clinical Pathology. 55(4): 298-304.

Ohkawara T, Takeda, H, Miyashita K et al. (2005) Regulation of toll-like receptor
4 expression in mouse colon by macrophage migration inhibitory factor.
Histochem. Cell Biol. 9: 1-8.

Vachon PH, Simoneau A, Herringgillam FE and Beaulieu JF. (1995) Cellular
Fibronectin Expression Is Down-Regulated at the Messenger-Rna Level in

Differentiating Human Intestinal Epithelial-Cells. Experimental Cell Research.
216(1): 30-34.

Gradl D, Kuhl M and Wedlich D. (1999) The Wnt/W g signal transducer beta-
catenin controls fibronectin expression. Molecular and Cellular Biology. 19(8):
5576-5587.

Takayasu H, Horie H, Hiyama E et al. (2001) Frequent deletions and mutations of
the beta-catenin gene are associated with overexpression of cyclin D1 and
fibronectin and poorly differentiated histology in childhood hepatoblastoma.
Clinical Cancer Research. 7(4): 901-908.

deGiorgio-Miller AM, Trehame LJ, McAnulty RJ et al. (2005) Procoltagen type I

gene expression and cell proliferation are increased in lipoderrnatoscterosis. British
Journal of Dermatology. 152(2): 242-249.

131

 

CHAPTER V.

BLACK BEANS AND SOY FLOUR REDUCE INF LAMMATORY

BIOMARKERS AND RESTORE COLONOCYTE DIFFERENTIATION

DURING AOM-INDUCED COLON CARCINOGENESIS IN RATS

132

_A_. ABSTRACT.

We have recently demonstrated that that dietary suppression of carcinogen-
induced colon cancer by beans was associated with modulation of mucosal genes
involved in cellular kinetics, energy metabolism, and innate immunity in rats.
Additionally, we identified sPLA2 as a potential modifier gene in this model, being
induced by AOM administration and suppressed by bean-feeding. sPLA2 is involved in a
variety of processes including anti-microbial defenses and inflammation. Therefore, this
study was conducted to further explore the involvement of this protein in colon cancer by
addressing whether induction of sPLA2 was associated with alterations in epithelial
cellular kinetics and immune responsiveness during the early stages of AOM-induced
colon carcinogenesis. Male F344 rats were obtained at 3 weeks of age and placed on a
control (AIN) diet. At 4 and 5 weeks of age, animals were administered subcutaneous
injections of AOM (n=60, 13 mg/kg) or saline (n=20). Beginning at 6 weeks of age, rats
injected with AOM were separated into one of three dietary treatments (AIN, black beans
(BB), or soy flour (SF)). Animals were sacrificed at 21 weeks of age and a section of the
medial half of the distal colon was processed to assess changes in epithelial proliferation,
sPLA2, and macrophage infiltration by immunohistochemistry. Rats injected with AOM
and fed the control diet (AIN) exhibited a higher percentage of proliferative cells in the
middle crypt compartment compared to rats fed BB, SF, or control-fed animals not
administered carcinogen. A positive correlation was found between sPLA2
immunoreactivity and the zone of proliferative cells. Macrophage infiltration was also
modestly higher in animals fed the control diet and administered AOM, suggesting a

generalized effect of bean diets on reducing colonic inflammation. We concluded that

133

early induction in sPLA2 is related to hyperplastic changes in colonic mucosa. The
ability of beans to suppress colon cancer is associated with modulating early biomarkers

of inflammation, potentially through enhancing cellular differentiation.

_l_3_. INTRODUCTION.

Dietary habits are strongly associated with the incidence of and mortality from
colorectal cancer (CRC) (1,2). Several experimental studies conducted in animals have
demonstrated that both dry beans and soy flour inhibit carcinogen-induced colon cancer
(3-8). Using microarrays, we recently reported that dietary suppression of colon cancer
in rats was associated with modulation of mucosal genes involved in cellular kinetics,
energy metabolism, and innate immunity (CHAPTER IV). More specifically, we
identified group IIA secretory phospholipase A2 (sPLA2), a pro-inflammatory protein, as
a potential target for CRC inhibition by beans. sPLA2 is involved in a variety of
processes, including anti-microbial defenses (9), sustained arachidonic acid release from
membranes (10), and activation of macrophages and neutrophils (1 1-13). The protein is
constitutively expressed in gland cells of some tissues including lacrimal, prostatic, and
seminal glands and in Paneth cells of the small intestine, suggesting anti-microbial
activity is important for host defenses (14). However, induction of sPLA2 in several
disease states including multi system organ failure, cardiovascular disease, ulcerative
colitis, and cancer indicates sPLA2 may perpetuate inflammation, likely through catalytic
production of lipid mediators (14-20).

In humans and in animals, sPLA2 is normally expressed at low levels in the
colon. Kennedy et al. (21) reported elevated levels of mRNA for sPLA2 in tumors from

humans with familial adenomatous polyposis (FAP), who have inherited susceptibility to

134

colon cancer development. Edhemovic et al. (22) also demonstrated a stronger
immunoreactivity of sPLA2 in the peritumoral mucosa than sites more distal to or within
the colon tissue. Additionally, in animals, administration of carcinogens to initiate colon
cancer enhances sPLA2 mRNA and protein expression (23-24). These results suggest a
role for sPLA2 in tumorigenesis, although the mechanisms involved have not been
elucidated. Igemagi et al. (23) found a correlation between sPLA2 activity and
production of the prostaglandins PCB; and 6-keto PGF; in colonic mucosa of animals
treated with AOM. PGE2 has been associated with promotion of colon cancer by
stimulating epithelial proliferation and inhibiting apoptosis (25-27). Additionally, tumor
tissue is heavily infiltrated with immune cells, and in vitro, sPLA2 stimulates superoxide
and nitric oxide release from neutrophils and in macrophages primed with the bacterial
antigen lipopolysaccharide (LPS) (ll-13). Thus, inability to suppress sPLA2 may
promote tumorigenesis indirectly by stimulating inflammatory cells to release by-
products, which can result in epithelial damage. The focus of the current investigation
was to determine if AOM-induced changes in sPLA2 expression were related to
alterations in colonic epithelial kinetics in rats and whether these changes could be
attenuated by bean-feeding. Macrophage infiltration in colonic tissue was also measured

to assess generalized dietary differences in inflammation.

_C_. MATERIALS AND METHODS.
Animal care and experimental diets.
This study was conducted in accordance with procedural guidelines of the

Michigan State University Committee on Animal Use and Care. Three-week old male

135

 

Fischer (F344) rats (n=90) were obtained from Harlan Sprague-Dawley (Indianapolis,
IN) and housed in plastic cages (2-3 rats/cage) in temperature (23°C d: 2°) and humidity
(40-60%) controlled rooms with a 12 hour light/dark cycle. Animals were observed daily
for health status and body weights were monitored on a weekly basis. Throughout the
experiment, animals had free access to food and house distilled water.

The experimental diets utilized in this study have previously been described in
detail (CHAPTER IV). Briefly, three diets were formulated based on the AIN-93G
rodent diet and contained 1) casein (AIN), 2) cooked, dried, ground black beans (BB), or
3) defatted soy flour (SF; Archer Daniels Midland; Decatur, IL) as the protein source.
All diets contained approximately 18.9% (wt/wt) total protein, 11.3% fiber, and 16.7%
fat (lard: soybean oil mixture).

Experimental procedures.

Animals were fed the control (AIN) diet upon arrival, and after a one week
acclimatization period, were injected once per week for two weeks with 13 mg/kg of
azoxymethane (AOM) (Sigma Chemical Co., St. Louis, MO) prepared in saline (n=60) or
saline (SHAMs, n=20). One week following injections (7 weeks of age), animals treated
with AOM were randomly assigned to either continue on the AIN (control) diet or
receive experimental diets (BB, SF). Saline-injected animals were fed the control (AIN)
diet throughout the course of the experiment. At 21 weeks of age, animals were
sacrificed by affixation with carbon dioxide and exsanguination, and colons were
immediately excised, opened longitudinally, and rinsed in tap water to remove debris.
The colon was transected into proximal and distal segments, and a section (2 cm) from

the medial half of the distal colon was excised, pinned flat on cardboard and fixed in 10%

136

 

neutral buffered formalin (NBF, pH 7.4) for 4-6 hours. Tissues were then dehydraded in
graded ethanol-water baths, rinsed in xylene, and infiltrated with paraffin prior to
immunohistochemical procedures (see below).

Immunohistochemistry.

Dietary influences on colonic expression of PCNA, sPLA2, and CD68+
macrophages were examined by peroxidase biotin-streptavidin immunohistochemistry.
Anti-human monoclonal antibody against group HA PLA2 (sPLA2) was purchased from
Caymen Chemical Co. (Ann Arbor, MI) and anti-rat CD68 monoclonal antibody from
Ab-CAM, Inc. (Cambridge, MA). Anti-human monoclonal antibody against proliferating
cell nuclear antigen (PCNA) and secondary antibodies were purchased from DAKO
(Carpinteria, CA). The remaining reagents were obtained from Sigma Chemical Co. (St.
Louis, MO) unless otherwise indicated.

Four—micron thick sections of colon tissue were placed on poly-L-lysine coated
slides and affixed to the slides by drying at 58 °C for 2 hours, deparaffinized in xylene,
and rehydrated in ethanol-H20 with a final rinse in H20. For antigen retrieval, slides
were immersed in a 10 mM citrate buffer (pH 6.0) containing 0.05% Tween—20 and
placed in a steamer for 30 minutes at 92-95 °C. After cooling to room temperature (20
minutes), sections were treated with a 3% H202 solution for 10 minutes to block
endogenous peroxidase activity, followed by a 1% bovine serum albumin (BSA) solution
prepared in Tris-buffered saline (TBS; 10 mM tris(hydroxymethyl)aminomethane-
buffered saline, pH 7.4) for 30 minutes to reduce non-specific binding of antibody.
Sections were then incubated overnight at 4 °C with primary antibodies diluted 1:500 in

TBS buffer containing 1% BSA. After extensive washing, tissue sections were treated

137

 

with biotinylated rabbit anti-mouse immunoglobulins followed by peroxidase-conjugated
streptavidin at room temperature for 1 hour each. Antigen-linked peroxidase was
detected with the chromagen 3-3’-diaminobenzidine (DAB) diluted in a 10 mM
phosphate buffered saline solution (PBS, pH 7.2) containing 0.015% H302 for 5 minutes.
Tissues were then lightly stained in Gill’s hemotoxylin, blued in 0.3% ammonium water,
then dehydrated in ethanol-xylene baths before mounting with Permount. All antibody
incubations were performed in Shandon racks and sections were rinsed with 10 mM TBS
(pH 7.4) between steps.

Quantitative analysis of immunohistochemistry.

For quantification of PCNA labeled cells, a researcher blinded to treatments
evaluated 10-20 full-length crypts/animal. The total number of nuclei (PCNA+ and
PCNA") lining one side ofthe crypt and extending from the base of the crypt (cell 1) to
the lumen was recorded. Crypt height (average number cells/hemicrypt), labeling index
(number of PCNA+ cells/crypt height) and proliferation zone (highest labeled PCNA+
cell/crypt height) were then calculated and analyzed. Colonic staining of sPLA2 and
CD68+ were quantified using BioQuant Imaging Software (Nashville, TN) and a light
microscope (Nikon Optiphot-Z; Kanagawa, Japan) equipped with a color video camera
(Sony, Hyper HAD; New York, NY). For sPLA2 determination a 40X objective was
used. Individual crypts projected on the computer screen were traced with a cursor, and
the thresholded stain was measured. Due to the high degree of background with sPLA2
staining, only the deeper part of the stain near the basolateral surface was quantified. The
heights of each crypt, extending from the base to the lumen, were also measured and

output values calibrated with a micrometer. CD68+ macrophages were quantitated using

138

a 20X objective. The lamina propria between and below crypts was traced and the
positive stained area (total number of pixels) were quantified. Data are expressed as a
percentage of positive stained area (macrophages) in relation to the total surface area.
For each stain, 10 measurements/animal were taken.

Statistical analyses.

Statistical analyses for body weights and immunohistochemical data were
performed using the General Linear Model procedure of SAS (SAS Institute, Inc. Cary,
NC, Version 7.0) and when appropriate, least squares means (LSM) were compared using
the least significant difference method. Correlation and linear regression analysis were

performed using Microsoft Excel 2000.

Q RESULTS.
Body weights.

There was no significant effect on body weight gain between groups. The final
weight gain (g, LSM i SEM) per group was: AIN(SHAM) = 214 i 4.2, AIN(AOM)=
222 i 4.1, BB(AOM) = 216 i 4.1, SF(AOM) = 209 i 4.1.

PCNA and sPLA2 immunohistochemistry.

Results for PCNA proliferation and sPLA2 indices are presented in TABLE 1.
There was no effect of treatment on whole crypt labeling index (P = 0.07). However,
significant differences were detected between groups in the percent of labeled cells per
crypt compartment and in the proliferation zone (highest PCNA+ cell/crypt). Rats
injected with AOM and fed the AIN diet had a higher proliferation zone (P = 0.0005)
compared to rats fed BB or SF or the control (saline-injected) group. Similar treatment

effects were also evident for the percent of proliferative cells in the middle crypt

139

 

compartment and sPLA2 immunoreactivity (TABLE 1, P = 0.0001). sPLA2 was
localized primarily in the lower crypt compartment towards the basolateral surface
(FIGURE 1). Further, a highly significant correlation between sPLA2 and the
proliferation zone (r = 0.86, P = 0.0001) was detected indicating 74% of the differences
in P2 between groups were related, in this study, to differences in the amount of sPLA2
(FIGURE 2).

Presence of CD68+ macrophages in colonic mucosa.

There was a modest, although significant effect of diet on macrophage infiltration
into the lamina propria (P = 0.04). Consistent with sPLA2 data, rats fed the control diet
and injected with AOM had a higher presence of macrophages than either rats fed the BB
or SF diets or in control-fed, saline-injected animals. Group means were 0.68%, 0.81%,
0.62%, and 0.64% for AIN(saline), AIN(AOM), BB(AOM), and SF(AOM) groups,
respectively (FIGURE 3). Tumor tissue from a separate study was included as a positive
control (5.4%). As shown in FIGURE 4, a majority of macrophages were situated

immediately below the epithelial layer near the crypt lumen.

140

 

 

 

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FIGURE 1. Immunoreactivity for sPLA2 in colonic mucosa. (A)
AIN-saline-injected, (B) AIN(AOM-injected), (C) BB(AOM-
injected), and (D) SF (AOM-injected animals. Arrows indicate
sPLA2 stained areas at the crypt base. Images in the dissertation are

presented in color.

142

sPLA2 intensity/crypt length

 

3.0

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R2 = 0.7412

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0.0 7 r , -
0.40 0.45 0.50 0.55 0.60 0.65 0.70

PCNA proliferation zone (highest PCNA + cell/crypt)

FIGURE 2. Correlation between PCNA proliferation zone
(highest PCNA+ cell/crypt height) and sPLA2
immunoreactivity in colonic crypts. Using regression
analysis, a highly significant correlation was detected (r = 0.86,

P = 0.0001).

143

 

 

% Lamina Propria Area covered by

CD68+ macrophages

0.9%

 

 

 

0.8%

0.7%

 

 

 

0.6% a

0.5%

0.4% r

0.3% 4

0.2%

0.1% ‘

 

 

 

 

0.0% ~ .
AIN Saline- AIN AOM- BB AOM- SF AOM-
injected injected injected injected

 

FIGURE 3. Percent area of distal colonic lamina propria
covered by CD68 macrophages. Lamina propria and CD68+ areas
were quantitated using Bioquant sofiware. Superscripts denote
significant differences between groups (P = 0.04). Tumor tissue was

used as a positive control (5.4%, data not shown).

 

 

 

 

 

 

 

 

 

 

 

 

 

FIGURE 4. Immunoreactivity for C68+ macrophages in colonic mucosa.
(A) AIN-saline-injected, (B) AIN(AOM-injected), (C) SF (AOM-injected)
and (D) BB(AOM-injected) animals, (E) colon tumor. Arrows indicate

CD68+ stained areas. Images in the dissertation are presented in color.

145

Q DISCUSSION.

Chronic inflammation is strongly associated with colon cancer development, and
in previous investigations, sPLA2 was identified as a potential modifier of AOM-induced
colon cancer in rats. Specifically, expression of sPLA2 was induced by AOM
administration irrespective of dietary treatment, but much less so in bean diets, which
also inhibit colon cancer in this model. In the current study, we evaluated sPLA2 in
colon tissue by immunohistochemistry to determine if AOM induced changes in sPLA2
were related to alterations in epithelial kinetics and inflammatory responses in rats and
whether these changes could be attenuated by bean-feeding. We found that rats
administered AOM and fed the control (AIN) diet had a higher zone of proliferative cells
in colon crypts than rats fed beans or control (saline-injected) animals. In addition, there
was an increase in the proportion of PCNA-labeled cells in the middle crypt
compartment. We also detected elevated expression of sPLA2 in AIN-fed, AOM-
injected animals and determined that expression of sPLA2 correlated significantly with
the proliferative zone, suggesting an inverse relationship between differentiation and
sPLA2 immunoreactivity.

Epithelial production of mucin glycoproteins and trefoil peptides as well as tight
junction barriers normally constrain exposure of the epithelium and underlying lamina
prOpria to lumenal antigens (28). The localization of sPLA2 within in the lower crypt
regions detected in this study and previously by others (24) may be important in
transcriptional regulation of the gene, thereby contributing to inflammatory events when
mucosal integrity is impaired. The rat gene encoding sPLA2 contains binding sites for

the transcription factors CEBP and nuclear factor K B (NFKB) (29), with the latter often

146

increased during inflammation and cancer (30). Toll-like receptor 4 (TLR4), also
expressed at the crypt base (31), mediates NFKB activation and cytokine release in
response to the bacterial antigen lipopolysaccharide (LPS) (32). It has been demonstrated
in vitro that induction of colonocyte differentiation results in down-regulation of TLR4
receptor mRNA and reduced responsiveness to LPS (33). Although differentiated cells
are more exposed to lumenal antigens and therefore would be expected to have lower
proinflammatory function, these data would suggest that the ratio of differentiated to
undifferentiated colonocytes within the crypt may affect the duration and severity of
inflammation invoked following damage to the epithelium or as a result of poor barrier
function.

Consistent with this, we also detected significant differences, although modest, in
macrophage infiltration in rats treated with AOM and fed the control diet. Resident
macrophages underlying the epithelial surface have been proposed to play a role in
immediate microbial defenses (34), however monocytes can be recruited to sites of
inflammation through local production of chemokines (35). Infiltration of tumor-
associated macrophages (TAM) is frequently observed during colon cancer development,
and in humans, increases during tumor progression (36). Chronic activation of
macrophages has been associated with cancer promotion by producing growth and
angiogenic factors and contributing to tissue damage (36). Although changes in the
percentage of macrophages were small in this study, a higher presence of macrophages
and sPLA2 in AIN—fed, AOM-treated animals compared to bean-fed animals is consistent

with enhanced tumori genesis.

147

 

In conclusion, this study demonstrated that bean-based diets, which inhibit
experimental colon cancer, also reduce early biomarkers of inflammation in the colonic
epithelium. Both black-bean and soy flour-fed animals treated with the carcinogen AOM
had lower colonic sPLA2, proliferative indices, and macrophage infiltration into the
colonic mucosa as compared to animals fed a casein-based diet. Further, we were able to
demonstrate a positive and significant correlation between the levels of sPLA2 within
colonic crypts and the proliferative zone. These data suggest that bean-feeding reduces
early inflammatory events induced by AOM, which is inversely associated with cellular

differentiation.

148

L

10.

ll.

12.

LITERATURE CITED.

Ferlay J, Bray F, Pisani P, and Parkin DM. (2002) GLOBOCAN 2002: Cancer
incidence, mortality, and prevalence worldwide, IARC Press, Lyon, France,
http://www-depdb.iarc.fr/globocan/GLOBOframe.htm.

American Institute of Cancer Research/World Cancer Research Fund
(AICR/WCRF). ( 1997) Colon, rectum. In Food, Nutrition and the Prevention of
Cancer: A Global Perspective, BANTA Book Group, Menasha, WI, pp. 216-51.

Hughes JS, Ganthavom C, and Wilson-Sanders S. (1997) Dry beans inhibit

azoxymethane-induced colon carcinogenesis in F344 rats. Journal of Nutrition.
127: 2328-2333

Hangen LA and Bennink MR. (2003) Consumption of black beans and navy beans
(Phaseolus vulgaris) reduced axozymethane-induced colon cancer in rats. Nutrition
and Cancer. 44: 60-6

Bennink MR and Om AS. (1998) Inhibition of colon cancer (CC) by soy
phytochemicals but not by soy protein. F aseb Journal. 12(5): 3808.

Bennink MR, Om AS and Miyagi Y. (1999) Inhibition of colon cancer (CC) by soy

flour but not by genistin or a mixture of isoflavones. Faseb Journal. 13(4): A50-
A50.

Bennink MR, Om AS and Miyagi Y. (2000) Inhibition of colon cancer (CC) by
wheat bran, soy flour, and flax. Faseb Journal. 14(4): A217-A217.

Rondini EA and Bennink MR. (2002) Defatted soy flour, but not soy concentrate,

reduces azoxymethane-induced colon carcinogenesis. Journal of Nutrition. 132(3):
5898-5898.

Buckland AG and Wilton DC. (2000) The antibacterial properties of secreted
phospholipases A(2). Biochimica Et Biophysica Acta-Molecular and Cell Biology
ofLipids.1488(1-2): 71-82.

Murakami M, Kudo I and Inoue K. (1995) Secretory Phospholipases a(2). Journal
of Lipid Mediators and Cell Signalling. 12(2-3): 1 19-130.

Zallen G, Moore BB, Johnson J L et al. (1998) New mechanisms by which secretory
phospholipase A(2) stimulates neutrophils to provoke the release of cytotoxic
agents. Archives ofSurgery. 133(11): 1229-1232.

Back SH, Kwon TK, Lim Jh et al. (2000) Secretory phospholipase AZ-potentiated

inducible nitric oxide synthase expression by macrophages requires NF-kappa B
activation.Jlmmunol. 15:164(12): 6359-65.

149

 

l3.

14.

15.

l6.

17.

18.

19.

20.

21.

22.

23.

Baek SH, Yun SS, Kwon TK et al. (1999) The effects of two new antagonists of

secretory PLA2 on TNF, iNOS, and COX-2 expression in activated macrophages.
Shock. 12(6): 473-8.

Nevalainen TJ, Haapamaki MM and Gronroos JM. (2000) Roles of secretory
phospholipases A(2) in inflammatory diseases and trauma. Biochimica Et
Biophysica Acta-Molecular and Cell Biology of Lipids. 1488(1-2): 83-90.

Ogawa M. (1997) Group II phospholipase A(2) in neoplastic diseases.
Phospholipase A2. 24(200-204).

Haapamaki MM, Gronroos JM, Nunni H et al. (1999) Phospholipase A(2) in serum
and colonic mucosa in ulcerative colitis. Scandinavian Journal of Clinical &
Laboratory Investigation. 59(4): 279-287.

Partrick DA, Moore BB, Silliman CC et al. (2001) Secretory phospholipase A(2)
activity correlates with postinjury multiple organ failure. Critical Care Medicine.
29(5): 989-993.

Graff JR, Konicek BW, Deddens JA et al. (2001) Expression of group Ila secretory
phospholipase A2 increases with prostate tumor grade. Clinical Cancer Research.
7(12): 3857-3861.

Niessen HWM, Krijnen PAJ, Visser CA et al. (2003) Type II secretory
phospholipase A2 in cardiovascular disease: a mediator in atherosclerosis and
ischemic damage to cardiomyocytes? Cardiovascular Research. 60(1): 68-77.

Fahlgren A, Hammarstrom S, Danielsson A and Hammarstrom ML. (2003)
Increased expression of antimicrobial peptides and lysozyme in colonic epithelial

cells of patients with ulcerative colitis. Clinical and Experimental Immunology.
131(1): 90-101.

Kennedy BP, Soravia C, Moffat J et al. (1998) Overexpression of the nonpancreatic
secretory group II PLA(2) messenger RNA and protein in colorectal adenomas
from familial adenomatous polyposis patients. Cancer Research. 58(3): 500-503.

Edhemovic 1, $an M, Kljun A and Golouh R. (2001) Immunohistochemical
localization of group H phospholipase A2 in the tumours and mucosa of the colon
and rectum. European Journal of Surgical Oncology. 27(6): 545-548.

Ikegami T, Matsuzaki Y, Shoda J et al. (1996) Ursodeoxycholic acid (UDCA)

suppresses enhanced expression of group-II phospholipase-A2 (PLA2) on colonic
tissue of azoxymethane (AOM) treated rats. Hepatology. 24(4): 1599-1599.

150

 

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

Papanikolaou A, Wang QS, Mulherkar R, Bolt A and Rosenberg DW. (2000)
Expression analysis of the group IIA secretory phospholipase A(2) in mice with

differential susceptibility to azoxymethane-induced colon tumorigenesis.
Carcinogenesis. 21(2): 133-138.

Qiao L, Kozoni V, Tsioulias GJ et al. (1995) Selected Eicosanoids Increase the
Proliferation Rate of Human Colon-Carcinoma Cell-Lines and Mouse Colonocytes
in-Vivo. Biochimica Et Biophysica Acta-Lipids and Lipid Metabolism. 1258(2):
215-223.

Piazza GA, Rahm ALK, Krutzsch M et al. (1995) Antineoplastic Drugs Sulindac
Sulfide and Sulfone Inhibit Cell-Growth by Inducing Apoptosis. Cancer Research.
55(14): 3110-3116.

Boolbol SK, Dannenberg AJ, Chadbum A et al. (1996) Cyclooxygenase-2
overexpression and tumor formation are blocked by sulindac in a murine model of
familial adenomatous polyposis. Cancer Research. 56(11): 2556-2560.

Mayer L. (2003) Mucosal immunity. Pediatrics. 1 11(6 Pt 3): 1595-600.

Andreani M, Olivier JL, Berenbaum F et al. (2000) Transcriptional regulation of
inflammatory secreted phospholipases A(2). Biochimica Et Biophysica Acta-
Molecular and Cell Biology of Lipids. 1488(1-2): 149-158.

Karin M and Greten FR. (2005) NF kappa B: Linking inflammation and immunity

to cancer development and progression. Nature Reviews Immunology. 5(10): 749-
75.9.

Ortega-Cava CF, Ishihara S et al. (2003) Strategic compartmentalization of Toll-
like receptor 4 in the mouse gut. J Immunol. 15;170(8): 3977-85.

Yuan Q and Walker WA. (2004) Innate immunity of the gut: Mucosal defense in
health and disease. Journal of Pediatric Gastroenterology and Nutrition. 38(5):
463-473.

Lee SK, 11 Kim T, Kim YK et al. (2005) Cellular differentiation-induced
attenuation of LPS response in HT-29 cells is related to the down-regulation of

TLR4 expression. Biochem Biophys Res Commun. 18;337(2): 457-63.

Gewirtz AT and Madara JL. (2001) Periscope, up! Monitoring microbes in the
intestine. Nature Immunology. 2(4): 288-290.

Balkwill F and Mantovani A. (2001) Inflammation and cancer: back to Virchow?
Lancet. 357(9255): 539-545.

151

VI. SUMMARY AND CONCLUSIONS.

152

In conclusion, using the AOM-induced rodent model, this research provided a
molecular basis for epidemiological and experimental observations that beans inhibit
colon cancer. In the first two experiments, microarrays were utilized to identify global
changes in gene expression contributing to the molecular pathogenesis of AOM-induced
colon carcinogenesis in rats. These studies demonstrated that induction of genes
involved in inflammation and immune responses are involved in colon cancer
progression and some of these genes (sPLA2, NP defensin 3a, MHC II-related genes)
appear to explain abnormalities in normal mucosa permissive for tumorigenesis. Feeding
bean-based diets, which inhibited tumor formation by 60% in these animals, modulated
colonic expression of genes consistent with reduced proliferation (CDC2, cyclin B],
TOP2A, retinoblastoma), higher apoptotic indices (GADD 45a, CEACAMl), reduced
innate immune responsiveness (defensin 30¢, sPLA2, TLR4), and alterations in
extracellular matrix components (fibronectin 1, collagen lal). More specifically, bean-
feeding suppressed carcinogen-induced upregulation of defensin 3a, sPLA2, fibronectin,
and collagen lal. These data implied an association between genes involved in crypt
cytokinetics and inflammation to future colon cancer development. Therefore in the third
experiment, immunohistochemical techniques were employed to assess dietary
differences in proliferation indices, sPLA2, and macrophage infiltration during AOM-
induced colon carcinogenesis. It was demonstrated that bean-feeding reduced
carcinogen-induced alterations in the percentage of proliferating cells in the middle crypt
compartment, the proliferation zone, the amount of sPLA2 protein, and moderately
reduced infiltration of macrophages in colonic mucosa. Further, a positive correlation

was found between the proliferation zone and immunoreactivity for sPLA2.

153

Collectively, microarray and histochemical data imply that bean-diets inhibit
tumorigenesis by helping maintain a microanatomical “niche” within colon crypts.
Restraining the expansion of proliferative cells towards the lumen is closely related to
lower expression of innate defense, inflammatory, and extracellular matrix genes more
characteristic of crypt progenitor cells. Dietary agents that increase the proportion of
differentiated cells and/or reinforce the mucosal barrier may reduce exposure of

proliferative cells to lumenal contents and thereby limit bacterial-induced inflammation.

154

VII. FUTURE DIRECTIONS.

155

Colon cancer is the fourth most common cancer and second leading cause of
cancer mortality in the United States. Although colon cancer, if detected at an early
stage, has a good prognosis, education and accessibility to health care are barriers to
screening in the general population and therefore only 39% of cancers are detected this
early. Dietary habits are strongly associated with cancer risk, and this research lends
further support to epidemiological and experimental data that consumption of bean-based
diets inhibits colon cancer development. The finding that beans reduce early biomarkers
of colonic inflammation is also consistent with the inverse association of long-term
NSAID use on CRC risk. By demonstrating that diet can modulate genes in normal
mucosa permissive for tumorigenesis provides a rationale basis for clinical trials to
determine the true potential of beans to prevent colon cancer in humans. Further, it is
well recognized that individuals previously treated for colon cancer are at a 30% risk of
recurrence of the disease. This observation, termed a “field defect” or “field
cancerization” is proposed to be related to molecular abnormalities in areas surrounding
cancer tissue. Results form this study suggest that some of these abnormalities are
related to changes in cytokinetic, inflammatory, and extracellular matrix components that
alter differentiation programming along the colonic crypt. Confirming some of these
gene changes in humans, further identifying what causes these changes to occur, and
determining if bean consumption can reverse these changes would strengthen the
relationship of bean consumption on colon cancer inhibition and may provide useful

adjunct therapy for those at risk.

156

 

VIII. APPENDICES.

157

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in-
AOM-induced colon cancers relative to normal colonic mucosa. 1

 

 

 

Gene
symbol Gene Title Tumor

WWW

Cd44 CD44 antigen 13
183151 lectin, galactose binding, soluble 1 13
learn] intercellular adhesion molecule 1 3.3
Itgal integrin alpha 1 3.0
61391 glypican l 2.5
Alcam activated leukocyte cell adhesion molecule 2.2
Glbl gap junction membrane channel protein beta 1 0.5
C1dn3 claudin 3 0-5
(3630311110 CEA-related cell adhesion molecule 10 0.3
Thb54 thrombospondin 4 0.2
II. Cytoskeleton, microtubule processes, structural filaments

Tubal alpha-tubulin 9.6
Cnn3 calponin 3, acidic 6.8
T3813 transgelin 6.4
Vim vimentin 6. 1
Acth actin, gamma 2 5.7
--- similar to tubulin, beta (predicted) 4.2
P183 plastin 3 (t-isoforrn) 32
5311111 stathmin 1 2.9
--- similar to type xv collagen (loc298069), mrna 2.8
KIN-13 keratin complex 1, acidic, gene 18 2.4
Cnpl Cyclic nucleotide phosphodiesterase 1 2.3
TCtCXl t-complex testis expressed 1 2.3
Tpml tropomyosin 1, alpha 2.3
Cf“ cofilin 1 2-2
Pdlim3 actinin alpha 2 associated lim protein 2.2
Myolb myosin ib 2-0
TubbS tubulin, beta 5 2.0

 

158

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) '

 

Gene
symbol Gene Title Tumor
WWW
--- myosin regulatory light chain 2.0
Myola myosin, heavy polypeptide-like 0.4
K1121 cytokeratin 21 0.2
--- similar to actinin, alpha 2 (loc291245), mrna 0.1
WEN—4012mm
Srd5a2 steroid 5-a1pha-reductase 2 11
--- udp glycosyltransferase 1 family polypeptide a2 4.3
Mgstl microsomal glutathione s-transferase 1 4.0
pr2 glutathione peroxidase 2 3.4
Cybb endothelial type gp91-phox gene 3.3
th xanthine dehydrogenase 2.9
Prdxl peroxiredoxin 1 2.6
Nqol nad(p)h dehydrogenase, quinone 1 2.3
Gsth glutathione s-transferase, mu 5 2.2
--- metallothionein 2.2
Pam peptidylglycine alpha-amidating monooxygenase 0.5
Cbe cytochrome b5 05
Cyp2j9 cytochrome p450 monooxygenase 0.5
Cbrl carbonyl reductase l 0.5
Tst thiosulfate sulfurtransferase 0.5
--- udp glycosyltransferase 1 family, polypeptide 36 0.4
Akr7 a3 aldo-keto reductase family 7, member A3 (aflatoxin aldehyde
reductase) 0.4
Natl n-acetyltransferase 1 (arylamine n-acetyltransferase) 0.4
Cyp3a13 cytochrome p450 3a9 0.4
Gstml glutathione s-transferase, mu 1 0.2
Nat2 n-acetyltransferase-Z 0.2
Cyp2d10 0.1

 

cytochrome p450 2d]

159

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) I

 

Gene
symbol Gene Title Tumor

LEM—£48m
Tgrnl transglutaminase 1 24
--- protease, serine, 22 (predicted) 6.1
--- lysyl oxidase 5.4
Pd84b phosphodiesterase 4b 5.1
P1331323 phosphatidate phosphohydrolase type 2a 3.6
--- Pctaire2 3.3
Apexl apurinic/apyrimidinic endonuclease 1 3.2
___ UDP-GalzbetaGlcNAc beta 1,4—galactosyltransferase,

polypeptide 1 3.1
Siatl sialyltransferase 1 2.7
Pa f ah 1 b2 Sigieieztfctivatmg factor acetylhydrolase alpha 2 subumt (paf ah 2.3
--- T Clo-like Rho GTPase 2.2
Prm t3 protein arginine n-methyltransferase 3(hnmp methyltransferase

s. cerevisiae)-1ike 3 2.2
--- protein kinase N3 (predicted) 2.1
--- phenylalanine-tRNA synthetase-like, alpha subunit (predicted) 2.0
Galnt5 udp-galnaczpolypeptide n-acetylgalactosaminyltransferase t5 0.5
Futl fucosyltransferase 1 0.5
Ctbs di-n-acetylchitobiase 0.5
Uael udp-n-acetylglucosamine-Z-epimerase/n—acetylmannosamine

kinase 0.5
Ckmtl creatine kinase, mitochondrial 1, ubiquitous 0.4
Gda guanine deaminase 0.4
Bpntl 3(2),5-bisphosphate nucleotidase 0.4
--- dimethylarginine dimethylaminohydrolase 1 0.4
Fthfd 10-fonnyltetrahydrofolate dehydrogenase 0.3
Ade3 adenosine monophosphate deaminase 3 0.2
Cd38 CD38 antigen 0.2
Ca4 carbonic anhydrase 4 — 0.2

 

 

 

160

 

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) '

 

 

 

 

161

Gene
symbol Gene Title Tumor

V. Other
--- Thymus cell antigen 1, theta 28
M813 matrix gla protein 16
HSPbl heat shock 27kda protein 1 12
Gbp2 guanylate binding protein 2, interferon-inducible 11
R6838 pancreatitis-associated protein 3 8.6
___ S779OO myosin regulatory light chain isofonn C [rats, Sprague-

Dawley, new-bom, heart ventricle, mRNA, 1008 nt] 8.3
Serpinhl serine (or cysteine) proteinase inhibitor, clade h, member 1 7.5
--- similar to nedd4 ww binding protein 4 (100311676), mrna 6.8
C3103 calcitonin/calcitonin-related polypeptide, alpha 6.4
Serpinel serine (or cysteine) proteinase inhibitor, member 1 6.1
Serpingl serine (or cysteine) proteinase inhibitor, clade g (c1 inhibitor),

member 1, (angioedema, hereditary) 5.5
AbiZ abl-interactor 2 5.4
--- interferon induced transmembrane protein 3-like 5.2
Lgals3bp peptidylprolyl isomerase c-associated protein 4.4
Caps caZ+-dependent activator protein 4.4
ch2 reticulocalbin 2 4.2
--- similar to gOSZ-like protein (loc289388), mrna 4.0
NUP54 nucleoporin p54 3.5
--- centaurin, beta I (predicted) 3.5
510034 8100 calcium-binding protein a4 3.4
Anxa6 annexin vi 3.1
Eif2b3 eukaryotic translation initiation factor 2b, subunit 3 (gamma,

58kd) 2.9
LOC257646 fem-domain—containing protein 163scii 2.9
--- similar to deoxycytidyl transferase (loc316344), mrna 2.7
ngal high mobility group at-hook 1 2.7
Emd emerin 2.7

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) ‘

 

 

Gene
symbol Gene Title Tumor

V. Other
me31 pmf32 protein 2.6
Anan annexin 5 2.6
--- immediate early response 3 2.5
--- similar to myelin protein zero-like 1; protein zero related

(loc360871), mrna 2.4
Ehd4 pincher 2.4
--- vacuole membrane protein 1 2.4
H5903 heat shock protein 1, alpha 2,4
--- similar to mcdc47 (100288532), mrna 2.3
PVR tumor-associated antigen 1 2.3
--- notchl -induced protein 2.3
NUdC nuclear distribution gene c homolog (aspergillus) 2.2
Sepwl selenoprotein w, muscle 1 2.1
Eefl 31 eukaryotic translation elongation factor 1 alpha 1 2.1
Serpian serine (or cysteine) proteinase inhibitor, clade b, member 5 2.1
Anva annexin A2 2.0
--- heat shock 90kDa protein 1, beta 2.0
Stipl stress-induced-phosphoprotein 1 (hsp70/hsp90-organizing

protein) 2.0
--- similar to expressed sequence au040575 (loc288003), mrna 2.0
CCt3 chaperonin containing TCPI, subunit 3 (gamma) 2.0
--- sic-interacting ankyrin—containing protein 0.5
Kail kangai 1 0.5
R65P18 regulated endocrine-specific protein 18 0.5
--- similar to mitochondrial ribosomal protein s6 (loc288253), mrna 0.4
Nmu neuromedin 0.4
M1163 mucin 3 0.4
T801112 trans-golgi network protein 1 0.3
Insigl insulin induced gene 1 0.3

162

 

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) '

 

 

Gene
symbol Gene Title Tumor
V. Other
Uncl 19 unc-l 19 homolog (c. elegans) 0.3
Calb3 calbindin 3 0.3
229072cds rnmucinr r.norvegicus (sprague dawley) mrna for
m mucin 0.2
--- endothelin 2 0-2
GP2 secretory (zymogen) granule membrane glycoprotein ng 0.2
--- m81920 ratintmuc rat mucin-like protein mrna 0.1
VI. RNA processing
Hnrpal heterogeneous nuclear ribonucleoprotein a1 3.9
heterogeneous nuclear ribonucleoproteins methyltransferase-like
Hrmt112 . .
2 (s. cerevrsrae) 3.4
SMNI survival motor neuron 3.3
3813 sjogren syndrome antigen b 2.3
APObCCI apolipoprotein b editing complex 1 0.3
VI]. Signal Transduction
--- tumor-associated calcium signal transducer 2 31
Pdgfra platelet derived growth factor receptor, alpha polypeptide 8.3
FStll follistatin-like ' 8.0
Ptprd protein tyrosine phosphatase, receptor type, d 7.9
Tn frs f1 1b tumor necrosrs factor receptor superfamily, member 11b
(osteoprotegenn) 6.4
Axin2 axin2 5.6
Ar gbp2 arg/abl-interacting protein argbp2 5.4
Ednrb endothelin receptor type b 4.7
Ptpro protein tyrosine phosphatase, receptor type, c 4.1
Ltbpl lanc (bacterial lantibiotic synthetase component c)-like 1 3.7
mitogen activated protein kinase kinase kinase kinase 1
--- (predicted) 3.6
Ptpro protein tyrosine phosphatase, receptor type, 0 3.4
Stk39 serine threonine kinase 39 (ste20/spsl homcflpg, yeast) 3.1

 

163

 

 

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) '

 

Gene
_zan—_._Eflflfi____m£
WM
Ptpre protein tyrosine phosphatase, receptor type, epsilon polypeptide 3.1
Pr 1‘82 protein kinase, cgmp- dependent, type ii 2.5
A3132 arrestin, beta 2 2.2
CSI'P2 cysteine-rich protein 2 2.2
Map2k6 mitogen-activated protein kinase kinase 6 0.5
Add3 adducin 3, gamma 0.5
--- protein tyrosine phosphatase, non-receptor type 18 (predicted) 0.5
Viprl vasoactive intestinal peptide receptor 1 0.5
Glgl selectin, endothelial cell, ligand 0.4
Chnl chimerin (chimaerin) l 0.4
Synng synaptogyrin 2 0.4
ng epidermal growth factor 04
Itpka inositol 1,4,5-triphosphate 3-kinase 0.3
Prkca protein kinase c, alpha 0.3
___ tumor necrosis factor (ligand) superfamily, member 13

(predicted) 0.3
Guca2a guanylate cyclase activator 2a 0.3
Csrp2 cysteine and glycine-rich protein 2 0.3
Duspl protein tyrosine phosphatase, non-receptor type 16 0.3
Bmp2 bone morphogenetic protein 2 0.2
Pde2a phosphodiesterase 2a, cgmp-stimulated 0.2
PC1653 phosphodiesterase 5a, cgmp-specific 0.2
Adora2b adenosine a2b receptor 02
Ndrg2 n-myc downstream-regulated gene 2 0.2
--- bone morphogenetic protein 3 0.1
PM“ phospholipase c, delta 1 0.1
Rasgrpl ras guanyl releasing protein 1 0.1
Sgk serum/glucocorticoid regulated kinase 0.1

 

164

 

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) I

 

Gene

WWW
M581 melanocyte-specific gene 1 protein 7.5
A5012 achaete-scute complex homolog-like 2 (drosophila) 6.3
335131 brain abundant, membrane attached signal protein 1 5.9
Id3 inhibitor of dna binding 3, dominant negative helix-loop-helix

protein 5.3
LhXZ lim homeobox protein 2 4.7
Cebpd CCAAT/enhancer binding protein (C/EBP), delta 2.8
Nr433 nuclear receptor subfamily 4, group a, member 3 2.5
--- transcription factor E2a 2.5
NOtChl notch gene homolog 1, (drosophila) 2.5
RUVbll ruvb-like protein 1 2.4
--- nucleolin 2.3
F1112 four and a half lim domains 2 2.2
--- tbp-interacting protein 120a 2.2
Mcmd6 mini chromosome maintenance deficient 6 (s. cerevisiae) 2.1
U33883 p105 coactivator 2.0
Ratireb iron-responsive element-binding protein 0.5
Nfldl nuclear receptor subfamily 1, group d, member 1 0.5
Amtl aryl hydrocarbon receptor nuclear translocator-like 0.5
Vdr vitamin d receptor 0.4
Egr2 early growth response 2 0.4
Foqu hnf-3/forkhead homolog-1 0.4
Nr4a2 nuclear receptor subfamily 4, group a, member 2 0.4
Nr3c2 nuclear receptor subfamily 3, group c, member 2 0.3
K1f4 kruppel-like factor 4 (gut) 0.3
Tth thyroid hormone receptor beta 0.3
erh4 nuclear receptor subfamily 1, group h, member 4 0.2
del synaptotaflin 4 0.1

 

 

 

165

 

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) '

 

 

 

 

 

 

 

166

Gene
symbol Gene Title Tumor
IX. EST, function unknown
--- similar to type i hair keratin 6 (100287698), mrna 54
Rat mixed-tissue library Rattus norvegicus cDNA clone
--- rx00304 3', mRNA sequence [Rattus norvegicus] 36
--- transcribed sequences 18
r0_AA875531 UI-R-EO-cv-f-lZ-O-UI.sl Rattus norvegicus
--- cDNA, 3' end /clone=UI-R-E0-0v-f-12-0-UI /clone_end=3'
/gb=AA875531 /gi=2980479 /ug=Rn.256 llen=433 10
similar to osteoblast specific factor 2 precursor (loc361945),
--- mrna 9.9
Tmeffl Rat mixed-tissue library Rattus norvegicus cDNA clone
rx00133 3', mRNA sequence [Rattus norvegicus] 8.8
--- transcribed sequences 8.2
___ similar to cornifin alpha (small proline-rich protein 1) (sprrl)
(100365848), mrna 6.2
--- similar to riken cdna 6720467003 (100297903), mrna 5.9
--- similar to traf2 and nck interacting kinase, splice variant 4
(100301363), mrna 5.9
--- transcribed sequences 5.9
--- transcribed sequences 5.4
--- transcribed sequences 4.6
--- similar to 10x1 protein (100315714), mrna 4.5
dd25 hypothetical protein 4.4
--- similar to ras association domain family 2 (100311437), mrna 4.1
--- transcribed sequence with strong similarity to protein sp:p00722
(e. coli) bgal_ecoli beta-galactosidase 4.1
--- similar to riken cdna c730007120 gene (100364396), mrna 3.8
--- similar to phospholipid scramblase 3 (100360549), mrna 3.8
--- similar to capg protein (100297339), mrna 3.7
transcribed sequence with moderate similarity to protein
--- sp:p00722 (e. coli) bga1_ecoli beta-galactosidase 3,4

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) I

 

Gene
symbol Gene Title Tumor

IX. EST, function unknown
m91234 rat vl30 element mrna /cds=unkn0wn /gb=rn91234

/gi=207671 /ug=rn.18005 /len=1132 3.4
--- similar to ab2-008 (100290270), mrna 3.3
--- similar to plakophilin 4 (p0071) (100295625), mrna 3.3
--- small proline-rich protein gene 3.3
___ transcribed sequence with moderate similarity to protein

sp:p00722 (e. coli) bgal_ecoli beta-galactosidase 3.2
--- transcribed sequences 3.1
--- dd3g2-3 mrna, partial sequence 3.1
--- 100363015 (100363015), mrna 3.1

similar to chromosome 14 open reading frame 50 (100299153),
--- mrna 3.1

Rat mixed-tissue library Rattus norvegicus cDNA clone
--- rx01696 3', mRNA sequence [Rattus norvegicus] 2.9
--- transcribed sequences 2.8
___ x62951mrna rnpbus19 r.norvegicus mrna (pbusl9) with

repetitive elements 2.7
--- transcribed sequences 2.6
--- similar to jtvl-pending protein (100288480), mrna 2.5
m Similar to T-cell receptor beta-chain 2.5
--- transcribed sequences 2.5
--- similar to riken cdna 6720485015 (100360639), mrna 2.5
--- similar to riken cdna 1500011h22 (100288667), mrna 2.3
--- transcribed sequences 2.3
--- similar to phosphoprotein enriched in astrocytes 15 (100364052),

mrna 2.3
--- transcribed sequences 2.3
_ Rat mixed-tissue library Rattus norvegicus cDNA clone

rx01427 3', mRNA sequence [Rattus norvegicus] 2.2

x054720ds#3 rnrep24r rat 2.4 kb repeat dna right terminal
--- region 2,2

 

167

 

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) '

 

 

Gene
symbol Gene Title Tumor
IX. EST function unknown
--- similar to hypothetical protein flj 14360 (100303792), mrna 2.2
___ x535 8lcds#3 mlined r.norvegicus long interspersed repetitive
dna containing 7 orf‘s 2.2
--- similar to 25 kda tk506-binding protein (100299104), mrna 2.2
--- similar to hypothetical protein (11 5wsu59e (100294810), mrna 2.2
transcribed sequence with moderate similarity to protein
--- ref:np_036191.1 (m.musculus) ca<2+>dependent activator 2.1
--- similar to flcsg27 (100292708), mrna 2.1
--- sideroflexin I (predicted) 2.1
similar to n-acetylglucosamine-6-sulfatase precursor (g6s)
--- (glucosamine-6-sulfatase) (100299825), mrna 2.1
--- similar to ga17 protein (100295975), mrna 2.1
/clone=RHEAC15 /clone_end=3' lgb=AA799526 /gi=2862481
' /ug=Rn.6351 /len=626 2.0
___ similar to myosin id (myosin heavy chain myr 4) (100289785),
mrna 2.0
--- transcribed sequences 20
--- similar to aspartyl aminopeptidase (100301529), mrna 2.0
--- transcribed sequences 2.0
--- transcribed sequences 0.5
MGC72616 unknown (protein for mgcz72616) 0.5
~-- similar to b0002216 protein (100313771), mrna 0.5
transcribed sequence with moderate similarity to protein
--- spqu3576 (h.sapiens) iqg2_human ras gtpase-activating-like 0.5
--- liver regeneration-related protein lrrg07 mrna, complete cds 0.5
--- similar to ms4a8b protein (100361733), mrna 0.5
--- similar to riken cdna 2700099019 (100302422), mrna 0.5
--- Similar to KIAA1592 protein 0.5
--- transcribed sequences 0.5
--- transcribed sequences 0.4

 

168

 

 

 

 

SUPPLEMENTARY TABLE 1. Supplemental list of global genetic alterations in
AOM-induced colon cancers relative to normal colonic mucosa. (continued) 1

 

Gene
symbol _ Gene Title Tumor

 

IX. EST, function unknown

similar to mitogen-inducible gene 6 protein homolog (mig-6)
(gene 33 polypeptide) (100313729), mrna 0.4

--- hypothetical 100290595 (100290595), ma 0.4

transcribed sequence with strong similarity to protein spzq15029
--- (h.sapiens) u581_human 116 kda u5 small nuclear

ribonucleoprotein component 0.4
--- x072660ds rrg33a rat mrna for gene 33 polypeptide 0.4
similar to bail-associated protein 1; ww domain-containing
--- protein 3; atrophin-l interacting protein 3 (100297463), mrna 0.4
--- transcribed sequences 0.3
--- transcribed sequences 0.3
--- similar to hypothetical protein MGC15606 0.3

r0_AA892248 EST196051 Rattus norvegicus cDNA, 3' end
--- /010ne=RKIA018 /clone_end=3' /gb=AA892248 /gi=3019127

/ug=Rn.2277 /len=587 0.3
___ Rat mixed-tissue library Rattus norvegicus cDNA clone

rx04959 3', mRNA sequence [Rattus norvegicus] 0.2
--- transcribed sequences 0.2

r0_AA892248 EST196051 Rattus norvegicus cDNA, 3' end
--- /clone=RKIA018 /clone_end=3' /gb=AA892248 /gi=3019127

/ug=Rn.2277 /len=587 0.2
--- similar to atp sulfurylase/aps kinase 2 (100294103), mrna 0.1
Rat mixed-tissue library Rattus norvegicus cDNA clone
--- rx01462 3', mRNA sequence [Rattus norvegicus] 0,1

 

 

I Data expressed as mean-fold change differences in tumor tissue relative to values for
non-neoplastic colon Lnucosa (n = 2-4/ group).

169

 

SUPPLEMENTARY TABLE 2. Supplemental list of genes differentially affected
by AOM treatment in distal colonic epithelium of male F344 rats. '

Saline- AOM-

 

Gene symbol Common Injected Injected
1. Cell cycle, cell growth and maintenance_, apoptosis

Cd53 0d53 antigen 1.0 a 2.2 b
--- clusterin 1.0 a 1.8 b
Sh3kbp1 sh3-domain kinase binding protein 1 1.0 a 1.5 b
Ns nucleostemin 1.0 a 1.4 b
E2f5 e2f transcription factor 5 1.0 a 1.3 b
c-fos c-fos oncogene 1.0 b 0.7 "
Apbb3 :r’nnyleqii 2103a (a4) precursor protein-binding, family 1.0 b 0.7 ,
Kitl kit ligand 1.0 b 0.7 8
Jun v-jun sarcoma virus 17 oncogene homolog (avian) 1.0 b 0.6 a
C amk2 d 3211::unV0almodulin-dependent protein kinase ii, 1.0 b 0. 6 a
II. Channel, transporters, & carriers

Apoe apolipoprotein e 1.0 a 1.3 b
Slc3a2 solute carrier family 3, member 2 1.0 ‘ 1.3 b
Slc4al solute carrier family 4, member 1 1.0 ‘ 1.2 b
Grina nmda receptor glutamate-binding chain 1.0 b 0.7 ‘1
Slc9a2 solute carrier family 9, member 2 1.0 b 0.7 "
Atplbl atpase na+/k+ transporting beta 1 polypeptide 1.0 b 0.7 ‘
III. Electron transpog oxidoreductase, detoxification

pr2 glutathione peroxidase 2 1.0 a 1.3 b
Cbrl carbonyl reductase 1 1.0 b 0.7 "
Dial diaphorase 1 1.0 b 0.7 ‘
Cyp2j9 cytochrome p450 monooxygenase 1.0 b 0.7 ‘
IV. Energy metabolism

Mel malic enzyme 1 1.0 a 1.4 b
Padi2 peptidyl arginine deiminase, type 2 1.0 a 1.4 b
Odcl omithine decarboxylase 1 1.0 ' 1.3 b

 

 

 

170

 

 

 

SUPPLEMENTARY TABLE 2. Supplemental list of genes differentially affected
by AOM treatment in distal colonic epithelium of male F344 rats. (continued) I

 

Gene symbOI Saline- AOM-
! J Common Injected Injected
IV. Energy metabolism

Fdftl famesyl diphosphate famesyl transferase 1 1.0 a 1.3 b

enoyl-coenzyme a, hydratase/3-hydroxyacyl

 

 

 

 

Ehhadh 1.0 b 0.7 "
coenzyme a dehydrogenase

Cdol cytosolic cysteine dioxygenase l 1.0 b 0.7 a

Pygb brain glycogen phosphorylase 1.0 b 0.7 a

V. Enzymes, other

LOC643OO cl-tetrahydrofolate synthase 1.0 a 1.4 b

Rab6 rab6, member ras oncogene family 1.0 a 1.3 b

Ca4 carbonic anhydrase 4 1.0 b 0.7 a

Ppm lb protem phosphatase lb, magnesrum dependent, beta 1.0 b 0.7 ,
isoform

Nu dt 4 diphosphomosrtol polyphosphate phosphohydolase 1.0 b 0.7 a
type 11

Comt catechol-o-methyltransferase 1.0 b 0.7 a

Natl n-acetyltransferase-2 1.0 b 0.6 a

Inpp4a inositol polyphosphate-4-phosphatase, type 1 1.0 b 0.5 a

VI. Nucleic acid bindipg, trapscription regulation

Baspl brain acidic membrane protein 1.0 a 1.5 b

ngb2 high mobility group box 2 1.0 a 1.4 b

Hnrpal heterogeneous nuclear ribonucleoprotein al 1.0 a 1.4 b

Nfia nuclear factor i/a 1.0 a 1.4 b

Gt f2 12 general transcnption factor 11f, polypeptide 2 (30kd 1.0 a 1.3 b
subumt)

Npml nucleophosmin l 1.0 a 1.3 b

Klf4 kruppel-like factor 4 (gut) 1.0 b 0.7 a

Nr5a2 nuclear receptor subfamily 5, group a, member 2 1.0 b 0.7 8

an4a hepatocyte nuclear factor 4, alpha 1.0 b 0.7 a

171

 

 

SUPPLEMENTARY TABLE 2. Supplemental list of genes differentially affected
by AOM treatment in distal colonic epithelium of male F344 rats. (continued) '

 

 

172

Gene symbol Saline- AOM'
Common . Inl'ected Inlected
VII. Signal transduction
Pde4b phosphodiesterase 4b 1.0 ‘ 1.4 b
Notch3 Notch 3 1.0 a 1.4 b
Aps 2:211:31); $.0ng gflsplflsm homology and src 1.0 , 1.3 b
Cxcr4 chemokine (C-X-C motif) receptor 4 1.0 a 1.3 b
Nmu neuromedin 1.0 ‘ 1.3 b
Oprsl opioid receptor, sigma l 1.0 " 1.3 b
Rgsl9 calcium/calmodulin-dependent protein kinase I 1.0 a 1.3 b
Cd81 CD 81 antigen 1.0 a 1.2 b
SstrZ somatostatin receptor 2 1.0 b 0.7 a
Erbb3 $213012: mixiwtic leukemia viral oncogene 1.0 b 0.7 ,
Pldl phospholipase D1 1.0 b 0.7 “
Plcdl phospholipase C, delta 1 1.0 b 0.7 "
KrasZ $8,263) rat sarcoma Viral oncogene homologue 2 1.0 b 0.7 ,
---- adrenergic receptor kinase, beta 1 1.0 b 0.7 “
Rabep2 rabaptin, RAB GTPase binding effector protein 2 1.0 b 0.7 '
Duspl dual specificity phosphatase 1 1.0 b 0.6 3
VIII. Other
th1 cardiotrophin l 1.0 a 1.5 b
Locl92269 pr-et2 encoded oncodevelopmental protein 1.0 a 1.4 b
U83883 p105 coactivator 1.0 a 1.4 b
Dnmll dynamin 1-1110 1.0 a 1.4 b
chcS :;:: 51:21:; :toempggesnting defective repair in 1.0 a 1.4 b
Pexl 1a pelagomsomal membrane protein pmp26p (peroxm- 1.0 a 1.4 b
Lgals3bp peptidylprolyl isomerase c-associated protein 1.0 a 1.4 b

 

 

SUPPLEMENTARY TABLE 2. Supplemental list of genes differentially affected
by AOM treatment in distal colonic epithelium of male F344 rats. (continued) '

 

Gene symbol Saline- AOM-
Common Injected Injected
VIII. Other
ch2 reticulocalbin 2 1.0 a 1.4 b
Naplll nucleosome assembly protein Mike 1 1.0 a 1.3 b
p47 p47 protein 1.0 a 1.3 b
SMNI survival motor neuron 1.0 a 1.3 b
Ssr3 trap-complex gamma subunit 1.0 a 1.3 b
Nolcl nucleolar phosphoprotein p130 1.0 a 1.3 b
Apbb3 cysteine string protein 1.0 b 0.7 a
Grina endothelin 2 1.0 b 0.6 a

IX. EST, fpnction unclassified

transcribed sequence with weak similarity to

---- protein ref:np_079050.1 (h.sapiens) hypothetical 1.0 a 1.8 b
protein flj21924 [homo sapiens]
similar to myosin id (myosin heavy chain myr 4)

a b
(100289785), mrna 1-0 1.6

transcribed sequence with weak similarity to
---- protein ref:np_006410.1 (h.sapiens) small 1.0 a 1.5 b
inducible cytokine b subfamily

similar to riken cdna 2610528023 (100288176),

1.0 a 1.5 b
mrna

---- similar to talin (100313494), mrna 1.0 a 1.4 b

---_ r0_h31217 est105044 rattus norvegicus cdna, 3' end 1 0 a 1 4 b
/clone=rpcaf34 /clone_end=3' /gb=h31217 ' '

_ similar to nadp+-specifi0 isocitrate dehydrogenase 1 0 a 1 4 b

(100293043), mrna ' °
similar to dna directed ma polymerase ii

_ polypeptide i; polymerase (ma) ii (dna directed) 1 0 a 1 4 b

polypeptide i (14.5kd); dna directed ma polymerase
ii 14.5 kda polypeptide (100292778), mrna

 

173

 

SUPPLEMENTARY TABLE 2. Supplemental list of genes differentially affected
by AOM treatment in distal colonic epithelium of male F344 rats. (continued) ‘

Gene symbol Saline- AOM-
Common Injected Iniected

IX. EST, function unclassified

rc_aa892400 est196203 rattus norvegicus cdna, 3'
---- end /0lone=rkiaq01 /clone_end=3' /gb=aa892400 1.0 a 1.4 b
/gi=3019279 /ug=rn.14755 /len=394

transcribed sequence with strong similarity to
---- protein ref:np_036526.2 (h.sapiens) prefoldin 2 1.0 " 1.4 b
[homo sapiens]

similar to riken cdna 1110032a17 (100362156),

 

 

1.0 a 1.4 b
mrna

---- similar to cu12 protein (100361258), mrna 1.0 a 1.3 b

---- dd6a4-2(5) mrna, partial sequence 1.0 a 1.3 b

____ similar to riken cdna 3110052n05 (100361351), 1 0 , 1 3 b
mrna ' °

---- transcribed sequences 1.0 a 1.3 1’

---- similar to 608 ribosomal protein l3-like 1 O a 1 3 b
(100287122), mrna ' °
r0_h31128 est104855 rattus norvegicus cdna, 3' end

---- /clone=rpcad04 /clone_end=3' lgb=h31128 1.0 a 1.3 b
lgi=976550 /ug==m.7211 /len=308

---- transcribed sequences 1.0 a 1.3 b

---- similar to riken cdna 2900010m23 (100361805), 1 0 a 1 3 b
mrna ° .

---- similar to interferon regulatory factor 7 (100293624) 1.0 a 1.3 b

---- transcribed sequences 1.0 a 1.3 b

transcribed sequence with moderate similarity to
protein pdbzllbg (e. coli) b chain b, lactose operon

a b
-_-- repressor bound to 21-base pair symmetric operator 1'0 1'3
dna, alpha carbons only
similar to hippocarnpus abundant gene transcript 1;
---- tetracycline transporter-like protein (100295398), 1.0 a 1.3 b
mrna

 

174

 

 

SUPPLEMENTARY TABLE 2. Supplemental list of genes differentially affected
by AOM treatment in distal colonic epithelium of male F344 rats. (continued) '

Gene symbol Saline- AOM-
Common Injected Injected

IX. EST, fwtion unclassified

similar to galactose-l-phosphate uridylyltransferase
--—- (gal-l-p uridylyltransferase) (udp-glucose——hexose- 1.0 a 1.3 b
l-phosphate uridylyltransferase) (100298003), mrna

---- transcribed sequences 1.0 a 1.3 b
similar to ubiquinol-cytochrome 0 reductase

—--— complex core protein 2, mitochondrial precursor 1.0 a 1.3 b
(complex iii subunit ii) (100293448), mrna
similar to protein translocation complex beta;

---- protein transport protein sec6l beta subunit 1.0 a 1.3 b
(100298068)

-_-- similar to unr-interacting protein (serine—threonine 1 O a 1 3 b
kinase receptor-associated protein) (100297699) ' '

---- similar to germinal histone h4 gene (100364721) 1.0 a 1.3 b
181 l36cds ratrpsZrla rattus norvegicus (strain r21) , b

__-- , , 1.0 1.3
rpsZrl preliminary dna, complete cds

---- transcribed sequences 1.0 a 1.3 b

---- transcribed sequences 1.0 a 1.3 b

---- similar to discs large homolog 7 (100289997), mrna 1.0 b 1.2 b

transcribed sequence with weak similarity to
---- protein ref:np_035569.1 (m.musculus) sry-box 1.0 b 0.7 3
containing gene 13 [mus musculus]

---- dd6a4-l mrna, partial sequence 1.0 b 0.7 a
---- line retrotransposable element 3 1.0 b 0.7 a
---- transcribed sequences 1.0 b 0-7 a

___ x535810ds#5 mlined r.norvegicus long interspersed 1 O b O 7 a
repetitive dna containing 7 orfs ' '

_--- similar to riken cdna 0610038110 gene (100317214), 1 0 b 0 7 a
mrna ' '

175

 

 

SUPPLEMENTARY TABLE 2. Supplemental list of genes differentially affected
by AOM treatment in distal colonic epithelium of male F344 rats. (continued) I

Gene symbol Saline— AOM-
Common Injected Injected

IX. EST, ftpiction unclassified

r0_aa859562 ui-r-eO-bv—b-03-0-ui.sl rattus
norvegicus cdna, 3' end /clone=ui-r-e0-bv-b-O3-0-ui

, . 1.0 b 0.7 a
/clone_end=3 /gb=aa859562 /g1=2949082
/ug=rn.268 /len=123
m131010ds ratlin4a rat long interspersed repetitive b ,
---- . 1.0 0.7
dna sequence hne4 (llrn)
rat mixed-tissue library rattus norvegicus cdna
---- clone rx00687 3', mrna sequence [rattus 1.0 b 0.7 "

norvegicus]

r0_aa894148 est197951 rattus norvegicus cdna, 3'
---- end /clone=rspar57 /clone_end=3' /gb=aa894148 1.0 b 0.6 a
/gi=3021027 /ug=m.15739 /len=448

similar to leucine-rich repeat-containing 8

b 3
(100311846), mrna 1-0 0.6

I Data expressed as mean-fold change differences standardized to the SHAM (saline-
injected) animals (n=12/ group). Superscripts denote significant main effects for
carcinogen treatment (P < 0.05).

176

 

SUPPLEMENTARY TABLE 3. Supplemental list of genes differentially

expressed by dietary treatment in distal colonic epithelium of male F344 rats. '

 

 

 

 

 

Gene

symbol Gene Title AIN BB SF
1. Cell cgcle,cellJrowth and maintenance, apoptosis
Pmp22 peripheral myelin protein 22 1.0 a 1.4 b 1.0 a
Btgl b-cell translocation gene 1 1.0 a 1.3 b 1.0 a
SlOOal 0 s-lOO related protein, clone 420 1.0 a 1.2 b 1.0 a
Ccnd2 cyclin d2 1.0 a 1.2 b 0.9 a
Bok b01-2-related ovarian killer protein 1.0 a 1.1 a 1.4 b
Btg3 b-cell translocation gene 3 1.0 a 1.0 a 1.4 b
Pcna proliferating cell nuclear antigen 1.0 “b 0.8 a 1.1 b
Rpa2 p32-subunit of replication protein a 1.0 ab 0.8 a 1.1 b
Cd025b cell division cycle 25b 1.0 b 0.8 a 1.0 b
11. Channel, transporters, & carriers
nyd4 :xyd domain-containing ion transport regulator 1.0 a 1.4 b 1.1 3
8101531 ffalr‘ltgfigfrniggrlf (Oligomptide 1.0 a 1.4 b 0.9 a
LOC64201 2-oxoglutarate carrier 1.0 a 1.2 ab 1.3 b
Calbl calbindin 1 1.0 8" 1.1 b 0.7 a
Abcb 6 :prrbgtcrhgg cassette, sub family b (mdr/tap), 1.0 a 1.0 3,, 1.3 b
3:228:02;Emittitisiitiiz‘ w w .
Abcb 1 a :pnbgfilpeg cassette, sub family b (mdr/tap), 1.0 a 1.0 a 2.0 b
Apob apolipoprotein b 1.0 a 1.0 a 1.3 b
Abcc3 :tlpnbgédigig cassette, sub-family c (cftr/mrp), 1.0 a 1.0 a 1.2 b
[11. Electron transporp oxidoreductase, detoxificatigi
Cat catalase 1.0 a 1.0 a 1.4 b
--- metallothionein 1.0 a 0.9 a 3.5 b
63th glutathione s-transferase, pi 2 1.0 ab 0.8 a 1.1 b

 

177

 

SUPPLEMENTARY TABLE 3. Supplemental list of genes differentially
expressed by dietary treatment in distal colonic epithelium of male F344 rats.
(continued) I

 

 

 

 

Gene

symbol Gene Title AIN BB SF
1V. Energy metabolism
Bach brain acyl-coa hydrolase 1.0 a 1.1 ab 1.5 b
Decrl 2,4-dienoyl coa reductase 1, mitochondrial 1.0 a 1.1 a 1.3 b
Cptla carnitine palmitoyltransferase 1, liver 1.0 a 1.1 a 1.3 b
--- farensyl diphosphate synthase 1.0 a 1.0 a 1.3 h
Crot carnitine o-octanoyltransferase 1.0 ab 0.9 a 1.3 b
Cth ctl target antigen 1.0 ab 0.9 a 1.2 b
Fpr fructose bisphosphatase 2 1.0 3" 0.8 a 1.1 b
V. Ejn_zymes, oth_e_r
--- gtp cyclohydrolase l 1.0 a 1.4 b 0.9 "
--- glutathione s-transferase, mu type 3 (yb3) 1.0 a 1.4 b 1.1 ab
Alasl aminolevulinic acid synthase 1 1.0 a 1.3 b 1.1 a
LOC81816 ubiquitin conjugating enzyme 1.0 a 1.3 b 1.2 3"
--- aldolase a 1.0 a 1.2 ab 1.6 b
chx gamma-glutamyl carboxylase 1.0 a 1.2 “b 1.4 "
Gcsl glucosidase 1 1.0 a 1.0 a 1.3 b

phosphoribosylpyrophosphate synthetase-

Prpsapl associated protein (39 kda) 1'0 ab 0'9 a 1'1 b
D dost 2;);lczl1tfini} eprlailpesphooligosacchande protem 10 ab 09 a 1.2 b
Psatl phosphoserine aminotransferase l 1.0 b 0.8 a 1.0 b
--- :yaplzgeaztszme guanine phosphoribosyl 1.0 b 0.8 a 1.0 b
NmeZ nucleoside diphosphate kinase 1.0 b 0.7 a 0.9 ab
der quinoid dihydropteridine reductase 1.0 b 0.7 a 0.9 b
H2afx transcribed sequences 1.0 b 0.7 a 1.0 b
Lck lymphocyte protein tyrosine kinase 1.0 b 0.7 a 0.9 ab
Ddx46 ma helicase 1.0 b 0.7 a 1.0 b
Fenl flap structure-specific endonuclease l 1.0 b 0.7 a 1.0 b

 

178

 

SUPPLEMENTARY TABLE 3. Supplemental list of genes differentially

expressed by dietary treatment in distal colonic epithelium of male F 344 rats.
(continued) I

 

 

 

 

 

Gene

symbol Gene Title AIN 33 SF
VI. Extracellular matrix, cell adhesiop, cytoskeletop
Timpl tissue inhibitor of metalloproteinase 1 1.0 a 1.1 b 0.9 3
Spare secreted acidic cysteine rich glycoprotein 1.0 b 1.0 b 0.5 a
Csrpl cysteine and glycine-rich protein 1 1.0 b 0.8 a 1.2 c
Myolb myosin ib 1.0 b 0.7 a 0.9 8
VII. Immune, defense, inflammation, stress
Hla-dmb rtl class ii, locus dmb 1.0 a 1.9 b 1.1 ab
Hla-dma rtl class ii, locus dma 1.0 a 1.5 b 1.1 3
VIII. Npcleic acid binding, transcription regulation
Ptbpl polypyrimidine tract binding protein 1.0 a 1.1 ab 1.3 b
Hesl hairy and enhancer of split 1 (drosophila) 1.0 a 1.1 a 1.3 b
nyb nuclear transcription factor-y beta 1.0 3" 0.9 a 1.1 b
Satb scaffold attachment factor b 1.0 ab 0.9 a 1.2 b
Nfia nuclear factor i/a 1.0 ab 0.9 a 1.3 b
HlfO hl histone family, member 0 1.0 b 0.9 a 1.1 C
3’
IX. Signal transduction
Mir16 membrane interacting protein of RGS16 1.0 a 1.3 b 1.0 a
Stk39 3:12;: threonine kinase 39 (ste20/spsl homolog, 1.0 a 1'1 ab 13 b
Plcb4 phospholipase 0, beta 4 1.0 a 0.9 a 1.2 b
P dg fra plaglilizreived growth factor receptor, alpha 1.0 b 0.9 ,b 0.7 ,
ng epidermal growth factor 1.0 b 0.7 a 1.0 b
Fgfr4 fibroblast growth factor receptor 4 1.0 b 0.7 a 1.0 b
PdeZa phosphodiesterase 2a, cgmp-stimulated 1.0 b 0.6 a 0.9 b

 

179

 

 

SUPPLEMENTARY TABLE 3. Supplemental list of genes differentially

expressed by dietary treatment in distal colonic epithelium of male F344 rats.
(continued) I

 

 

salivary prolillne-rich protein precursor

180

i

Gene

symbol Gene Title AIN 133 SF
X. Other
qutml sequestosome 1 1.0 a 1.4 b 1.2 ab
My d1 1 6 rlnlyseloid differentiation primary response gene 10 a 1.4 b 12 ab
Hpcall neural visinin-like ca2+-binding protein type 3 1.0 a 1.4 b 1.3 b
Muc3 mucin 3 1.0 a 1.4 b 1.1 ab
--- adipose differentiation-related protein 1.0 a 1.3 C 1.2 b
LgalsS lectin, galactose binding, soluble 8 1.0 a 1.3 b 1.1 ab
Aplp2 amyloid beta (a4) precursor-like protein 2 1.0 a 1.3 b 1.3 b
qulnl ubiquilin 1 1.0 a 1.3 b 1.1 ab
Uncl l9 un0-119 homolog (c. elegans) 1.0 a 1.3 b 1.1 a
--- line retrotransposable element 3 1.0 b 1.2 b 0.6 a
Canx calnexin 1.0 a 1.1 ab 1.3 b
Ftll ferritin light chain 1 1.0 a 1.1 b 0.9 a
Utm utrophin 1.0 a 1.1 a 1.6 b
--- annexin 1 1.0 b 1.0 b 0.7 a
Mlhl mismatch repair protein 1.0 a 0.9 a 1.2 b
Apbb3 :arrllyilliicll) b3: 1:121:42 precursor protein-binding, 1.0 a 0.9 a 1.4 b
--- heat shock protein precursor 1.0 ab 0.9 a 1.2 b
Pex6 peroxisomal biogenesis factor 6 1.0 3" 0.8 a 1.1 b
Fkbpl a fl(506-binding protein la 1.0 b 0.7 a 0.9 b
hr hairless 1.0 b 0.7 a 0.9 ab
XI. EST, function unclassified

transcribed sequence with weak similarity to

--- protein sp:p04280 (h.sapiens) prp1_human 1.0 a 1.7 b 1.7 b

 

SUPPLEMENTARY TABLE 3. Supplemental list of genes differentially

expressed by dietary treatment in distal colonic epithelium of male F344 rats.
(continued) I

 

 

symbol Gene Title AIN BB SF

XI. EST, fppction unclassified
rat mixed-tissue library rattus norvegicus cdna

--- clone rx04144 3', mrna sequence [rattus 1.0 a 1.6 b 1.8 b
norvegicus]

--- similar to riken cdna 2310046k01 (100311536), 10 a 1' 6 b 1.2 ,b
mrna

_-_ similar to riken cdna 0610006f02 (100366792), 10 a 1. 6 b 1. 6 b
mrna

--- transcribed sequences 1.0 a 1.4 b 1.2 "

--- parturition-related protein prp3 1.0 a 1.4 b 0.9 a

--- similar to nima-related protein kinase 1 0 a 1 4 b 1 4 b
(100299204), mrna ' ' °
similar to smooth muscle myosin phosphatase

-_- regulatory subunit homolog family member, 1 0 a 1 4 b 1 2 ab
maternal effect lethal mel-ll (110.6 kd) (mel- ' ° '
11) (100366002), mrna

--- transcribed sequences 1.0 a 1.4 ab 1.6 b

___ similar to hypothetical protein flj10579 1 0 a 1 3 b 1 1 a
(10031 1328), mrna ' ' °

--— similar to mkiaal737 protein (100314330), mrna 1.0 a 1.3 b 1.1 a

--- transcribed sequences 1.0 a 1.3 b 1.0 "
transcribed sequence with weak similarity to

—-- protein sp:p00722 (e. coli) bgal_ecoli beta- 1.0 a 1.3 b 1.1 ab
galactosidase

--- tumor rejection antigen gp96 1.0 a 1.3 b 1.5 b

p55 1.0 a 1.3 b 1.1ab

--_ similar to riken cdna 2210008a03 gene 1 0 a 1 2 ab 1 3 b
(100314438), mrna ' ' '
similar to endoplasmin precursor (endoplasmic
reticulum protein 99) (94 kda glucose-regulated

--- protein) (grp94) (erp99) (polymorphic tumor 1.0 a 1.2 ab 1.5 b

rejection antigen 1) (tumor rejection antigen
gp96) (100362862), mrna

181

 

SUPPLEMENTARY TABLE 3. Supplemental list of genes differentially

expressed by dietary treatment in distal colonic epithelium of male F344 rats.
(continued) I

 

 

Gene
symbol Gene Title AIN BB SF

XI. EST. ftpictiopppgllssifig
transcribed sequence with moderate similarity

--- to protein sp:p00722 (e. coli) bgal_ecoli beta- 1.0 a 1.1 a 1.4 b
galactosidase
similar to hypothetical protein flj21827 1 0 a 1 I ab 1 3 b
(100300675), mrna ' ' - '
u11071 mpabpr2 rattus norvegicus Sprague—

--- dawley polyadenylate-binding protein-related 1.0 b 1.0 b 0.8 a
protein mrna, 3' end

--- similar to sorting nexin 4 (100360725), mrna 1.0 a 1.0 a 1.3 b
similar to chromosome 10 open reading frame

--- 4; similar to putative acid phosphatase Q60] 1.] 1.0 b 0.9 “b 0.7 3
(100365458)

--- transcribed sequences 1.0 ab 0.9 a 1.1 b

--- transcribed sequences 1.0 b 0.8 a 1.0 b

--- r0_aa800849 est190346 rattus norvegicus cdna 1.0 b 0.8 “b 0.7 a

--- transcribed sequences 1.0 b 0.8 a 1.0 b
transcribed sequence with moderate similarity

--- to protein pirza41109 (h.sapiens) a41109 1.0 b 0.8 a 0.7 a
protein-tyrosine-phosphatase

-_- similar to n-terminal aceyltransferase l 1 0 b 0 8 a 1 1 b
(100310399), mrna ' i '
transcribed sequence with strong similarity to

--- protein sp:p00722 (e. coli) bgal_ecoli beta- 1.0 b 0.8 a 1.0 b
galactosidase
similar to acetyl coa transferase-like b a b

(100308100), mrna 1'0 0'7 1'0
similar to hypothetical protein flj10241 b a a

(100361520), mrna 1‘0 0'7 0'7
similar to hcv ns3-transactivated protein 1 b

--- . .7 a . a
(100299507), mrna 1 0 0 0 7

_-- Similar to transforming protein bmil - mouse 10 b 0.7 a 0.8 ,

(100307151)

182

 

SUPPLEMENTARY TABLE 3. Supplemental list of genes differentially

expressed by dietary treatment in distal colonic epithelium of male F344 rats.
(continued) I

 

 

Gene
symbol Gene Title AIN BB SF

XI. EST, functioppllgagifiLd

--- transcribed sequences 1.0 b 0.7 a 0.8 a

--- r0_aa892526 est196329 rattus norvegicus cdna 1.0 b 0.7 a 0.8 ab

Smcl 11 smc-like 1 (yeast) 1.0 c 0.7 a 0.8 b
similar to nedd4 ww binding protein 4 b a ,

--- (10031 1676) 1.0 0.7 0.7
x054720ds#2 rnrep24r rat 2.4 kb repeat dna b b a

-_- . . . 1.0 0.7 0.3
right term1nal region

--- transcribed sequences 1.0 b 0.7 a 0.8 ab

--- transcribed sequences 1.0 b 0.7 a 0.8 ab

--- erythrocyte protein band 4.1-like 3 1.0 b 0.7 a 1.1 b
similar to dual-specificity tyrosine-

--- phosphorylation regulated kinase 4 1.0 b 0.7 a 0.9 b
(100312721), mrna

-_- r0_aa866345 ui-r-aO-bm-a-04-O-ul.sl rattus 1.0 b 0.7 a 0.9 b
norvegicus cdna

--- similar to capg protein (100297339), mrna 1.0 b 0.6 a 0.8 ab

--- 100363015 (100363015), mrna 1.0 b 0.6 a 1.0 b
similar to protein tyrosine phosphatase 20 C a b

(100301333) 1.0 0.6 0.8
similar to hypothetical protein flj20531 b a b

--- (100303164) 1.0 0.6 0.9

--- similar to smc4 protein (100295107), mrna 1.0 b 0.6 a 0.8 a

-_- similar to expressed sequence au040575 1 0 b O 6 a 1 1 b
(100288003) ' ' '

hypothetical 100295337 (100295337), mrna 1.0 b 0.6 a 0.7 a
similar to rac gtpase-activating protein b a b
(100315298), mrna 1'0 0'5 0'8

--- r0_aa860039 u1-r—e0-bz-f-06-0-ui.52 rattus 1.0 b 0.5 a 0.7 b

norvegicus cdna

183

 

 

SUPPLEMENTARY TABLE 3. Supplemental list of genes differentially
expressed by dietary treatment in distal colonic epithelium of male F344 rats.
(continued) 1

Gene
symbol Gene Title AIN BB SF

XI. EST, flpiction unclassified

transcribed sequence with weak similarity to
___ protein ref:np_07l357.l (h.sapiens) l O b 0 4 a 0 9 b
hypothetical protein flj22794; kiaa1895 protein ' - .

[homo sapiens]
--- transcribed sequences 1.0 b 0.4 a 0.5 a

x62951mrna mpbus19 r.norvegicus mrna

b a ,
(pbusl9) with repetitive elements 1‘0 0'4 0-5

I Data expressed as mean-fold change differences standardized to the AIN group
(n=8/ group). Superscripts denote significant main effects for diet (P < 0.05).

184

 

 

 

 

 

      
  

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