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THE

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This is to certify that the

dissertation entitled

Dietary Modulation of Tumorigenesis and Gene
Expression in the MIN Mouse, A Genetic
Model for Human Colorectal Cancer

presented by

Mridvika

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in Human Nutrit ion

 

 

'- ’ C,
Major professor

Date March 28, 2001

 

MS U is an Affirmatiw Action/Equal Opportunity Institution 0-12771

 

 

LEBRARY

Michigan State
University

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 cJClRClDateDue.p65-p.15

DIETARY MODULATION OF TUMORIGENESIS AND GENE EXPRESSION
IN THE MIN MOUSE, A GENETIC MODEL FOR
HUMAN COLORECTAL CANCER
BY

Mridvika

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

2001

ABSTRACT

DIETARY MODULATION ON TUMORIGENESIS AND GENE EXPRESSION
IN THE MIN MOUSE, A GENETIC MODEL FOR
HUMAN COLORECTAL CANCER
By

Mridvika

Mutations in the adenomatous polyposis coli (Apc) gene have been identified in
human colorectal cancer and in familial adenomatous polyposis. The ApcMin (or Min)
mouse carries a germline mutation in the Apc gene and develops adenomas in the small
intestine and colon, making it an ideal model for the study of intestinal cancers.

The purpose of this dissertation was two-fold: 1) to examine the effects of three dietary
treatments (food restriction, feeding a defatted flax/whole-wheat-flour based diet, and
feeding a black bean-based diet) on tumor multiplicity and growth in small intestine and
colon in the Min mouse; 2) to provide, if possible, a molecular basis for the effects
observed. The non-steroidal anti-inflammatory drug (NSAID) sulindac was used as the
positive control.

The first study demonstrated that dietary treatments could alter intestinal
tumorigenesis. Tumor number in the small intestine decreased (P<0.05) from 76:58 in
the casein fed control group to 45:53 in the sulindac group (42% decrease), 5315.5 in
the flax/wheat group (30% decrease), 55i5-7 in the black bean group (28% decrease) and
48:99 in the diet restricted group (36% decrease). Sulindac reduced average small
intestine tumor diameter from 1.3 to 1.0 mm (p<0.01) while black bean and flax/wheat

diets increased tumor diameter (p<0.05). Colon tumor numbers did not differ

significantly among groups, but there was an increase in colon tumor size in mice fed
black bean and flax/wheat diets compared to the control group.

Since energy restriction in study one had the most dramatic impact on tumor
growth, the second study focused on changes in tumor number and size in Min mice after
3, 6 and 10 weeks of 30% energy restriction. Complimentary DNA arrays were used to
determine changes in expression of 1,200 mouse genes in small intestinal tumor tissue
from mice fed ad libitum or restricted in energy intake. The results indicate a significant
inhibition of tumor multiplicity and size in the small intestine after 3, 6 and 10 weeks of
energy restriction (p<0.05). In the colon, the changes in adenoma number and volume
caused by energy restriction were not significant. Of 1,200 genes studies, eight were
overexpressed (21.6 fold) and 13 were underexpressed (21.6 fold) in restricted mice in
comparison to control mice. In tumors from the energy restricted mice 30% percent of
the underexpressed genes were oncogenes and 50% of the overexpressed genes were
immune function proteins.

This research clearly demonstrated that energy restriction will inhibit small
intestine tumor growth in Min mice. Furthermore, tumors in mice fed fewer calories had
reduced expression of oncogenes and increased expression of genes related to immune

function.

This dissertation is lovingly dedicated to my family, Shakun and Sarban Brar, Ana
and Marty Vyakarnam, Enric and Rosa Maria, and my husband, Josep

ACKNOWLEDGEMENTS

I would like to extend my sincere gratitude and appreciation to my major
professor, Dr. Maurice Bennink for his incredible ideas, enthusiasm, and insight into this
work. He was a pillar of constancy during good times and bad, and I value his
generosity.

I thank Dr. Dale Romsos for his inspiring style of teaching, his ability to always
keep students on their toes, and his kindness. As a member of my guidance committee, he
always found time to inquire after the progress of my research despite a demanding
schedule.

Dr. Hord, member of my guidance committee breathed new life into my program
with fresh ideas and suggestions. I would like to thank him for the younger perspective
on my committee and his encouraging attitude.

I thank members of my guidance committee Drs. Schroeder and Bursian for their
valuable ideas, suggestions and unrelenting kindness.

I would like to thank Dr. Leslie Bourquin for access to his Min mouse colony for
experiments in Chapter 2. Sincere thanks are also extended to Dr. Timothy Zacharewski
and Mr. Mark Fielden for lending me the use of their lab and expertise for the DNA array
experiments. A special thanks goes to Mark for his time, technical expertise and
generosity.

Thanks to Ms. Shelli Pfiefer and Mr. Don Herrington for their efficiency, and
people in my lab, Laura Hangen, Elisabeth Rondini for their help at odd times.

My dear friends Dr. Anahita Mistry, 800 Young Kang and Dr. Arti Arora deserve

a special mention for keeping me grounded during my highs and afloat during my lows.

They were my confidants and sources of professional advice when I most needed it. My
friends across oceans were always supportive despite boundaries of time and space: Dr
Vanee Chonhenchob in Thailand, Dr. Ivy Hsu in Taiwan and Dr. Maria Marin-Martinez
in Spain. Through the beauty of their respective souls, they have taught me of humility
and patience, for which I will forever be grateful. Thanks to Micah Stowe, Shama Joseph
and Megan Waynar for all the fun Saturday nights that thankfully punctuated the
sometimes—unbearable drudgery of dissertation writing.

Finally, words cannot express the gratitude I feel towards my family. My parents
that acted as part parents, part therapists over long phone conversations and my sister for
always drawing my attention to my goals. My dear husband for his love and for making
me believe that it could be done. Their confidence in me, and love for me is the sap I

draw on as I stand erect and face the future.

vi

TABLE OF CONTENTS

LIST OF TABLES ........................................................................ x
LIST OF FIGURES ........................................................................ xi
CHAPTER 1. Literature Review ......................................................... 1
Diet and Colon Cancer ............................................................... 2
Genetic Basis of Colon Cancer ..................................................... 3
Colon Cancer and the Apc Gene ................................................... 4
Apc Mutations in Human Colon Cancers ......................................... 6
Rodent Models in Colon Cancer Research ....................................... 7
The Min Mouse ....................................................................... 8
Min Mouse in Nutrition Studies .................................................... 10
Non Steroidal Anti Inflammatory Drugs .................................. O ........ 13
Whole Wheat and Cancer ........................................................... 15
Flax and Cancer ...................................................................... 17
Black Beans and Cancer ............................................................ 18
Energy Restriction and Cancer .................................................... l9
Complimentary DNA Arrays ....................................................... 21
Literature Cited ................................................................................ 26

CHAPTER II. Inhibition of Intestinal Tumorigenesis in ApcMi“ Mice by Food

Restriction, Whole Wheat-Flaxseed Diet and Black Bean Diet .................... 34
ABSTRACT ................................................................................... 35
INTRODUCTION ............................................................................ 36
MATERLALS AND METHODS ........................................................... 40

vii

Experimental Design ................................................................ 40

Animal Housing ...................................................................... 40
Animal diets .......................................................................... 40
Tissue Preparation ................................................................... 42
Statistical Analysis ................................................................... 43
RESULTS ...................................................................................... 44
Growth ................................................................................ 44
Small Intestinal Adenomas ........................................................ 44
Colon Adenomas .................................................................... 48
DISCUSSION ................................................................................ 50
Literature Cited ............................................................................... 55

CHAPTER III. Energy Restriction Inhibits Tumorigenesis and Alters Gene

Expression in ApcMin Mice ............................................................... 60
ABSTRACT .................................................................................. 61
INTRODUCTION ........................................................................... 62
MATERIALS AND METHODS ........................................................... 65
Experimental Design ................................................................ 65
Animal diets .......................................................................... 65
Tissue Preparation .................................................................. 65
RNA Isolation ........................................................................ 67
cDNA Synthesis and Hybridization .............................................. 67
RESULTS ..................................................................................... 69

viii

Growth ............................................................................... 69

Small Intestinal Adenoma Number ............................................... 69
Small Intestinal Adenoma Diameter ............................................. 69
Colon Tumor Number ............................................................... 73
Colon Tumor Volume ............................................................... 73
Gene Expression ..................................................................... 76
DISCUSSION ................................................................................. 79
Literature Cited ................................................................................ 83

ix

LIST OF TABLES
CHAPTER I

Table 1. Studies investigating the effects of dietary factors on

tumorigenesis in the Min mouse ........................................ 11
CHAPTER II.
Table 1. Composition of the control, black bean and defatted
flax/whole-wheat diets .................................................... 41
Table 2. Overall gender effects in SI and colon adenoma
numbers and sizes in Min mice ......................................... 46
Table 3. Small intestinal tumor number and diameter in Min
mice fed control diet and treatment diets .............................. 47
Table 4. Colon tumor number and volume in Min mice fed the
control and treatment diets ............................................... 49
CHAPTER III.
Table 1. Composition of experimental semipurified diets fed
to control and energy restricted groups and their mean
daily food intake .......................................................... 66
Table 2. Over-expressed genes in tumor tissue of energy restricted
mice] ..................................................................... 77

Table 3. Under-expressed genes in tumor tissue of energy restricted

rmce .....................................................................

..... 78

LIST OF FIGURES
CHAPTER I

Figure 1. Multistep model for the development of colorectal
carcinogenesis ........................................................... 5

Figure 2. . Potential Apc functional domains and location of Apc mutations
in human colon cancer and Min mutation. (Adapted from

Shoemaker et al., 1997) ................................................ 9
CHAPTER H.
Figure 1. Weight gain of mice in treatment groups CT, FW, SL, BB and
FR .......................................................................... 45
CHAPTER 111.

Figure 1. Weight of ad libitum fed control mice and 30% energy
restricted mice over the 10 week study period ....................... 70

Figure 2. Small intestine adenoma numbers in ad libitum-fed control

mice and 30% energy restricted mice at 0, 3, 6 and 10 weeks
of treatment ................................................................ 71

Figure 3. Small intestine adenoma diameters in ad libitum-fed
control mice and 30% energy restricted mice at 0, 3, 6
and 10 weeks of treatment ............................................... 72

Figure 4. Tumor multiplicity (number of colon tumors/ number of
mice) in ad libitum fed control mice and 30% energy
restricted mice at 0, 3, 6 and 10 weeks of treatment. ............... 74

Figure 5. Colon tumor volumes for ad libitum-fed control mice

and 30% energy restricted mice at 0, 3, 6 and 10 weeks
of treatment. ............................................................. 75

xi

CHAPTER 1

LITERATURE REVIEW

LITERATURE REVIEW

This year nearly 552,200 Americans are expected to die of cancer—an average of
more than 1,500 people a day. Cancer is the second leading cause of death in the United
States, exceeded only by heart disease. In the United States, one of every four deaths is
from cancer. Colorectal cancers are the third most common cancers in men and women
with an estimated 93,800 new cases of colon cancer and 36,400 of rectal cancer in the
year 2000. The American Cancer Society has estimated 56,300 deaths (47,700 from
colon cancer, 8,600 from rectal cancer) for the year 2000, accounting for about 11% of all
cancer deaths. On a positive note, incidence rates for colon and rectal cancers have
declined significantly from 1992 tol996 (-2.1% per year). Research suggests that this
improvement may be due to early detection due to increased screening and polyp

removal, preventing progression of polyps to invasive cancers (ACS, 2000).

Diet and Colon Cancer

Colon cancer is one of the most common cancers in the Western world. Environmental
factors are the predominant causal factors as exemplified by a change in the incidence of
colon cancer when people emigrate from a low- to a high-incidence country (Haenszel
and Kurihara, 1968). It has been suggested that a diet high in energy, fat (Stemmerrnann
et al., 1984), and meat content (Enstrom, 1975) and low in fiber content (Burkitt. 1969:
Giovannucci et al, 1994), (i.e. a Western style diet) may be responsible (Kotake et al.,
1995). Conversely a diet high in fruits and vegetables is linked to a lower risk for

developing colon cancer (Hertog et al., 1996). Other dietary components reported to

influence colon cancer risk include micronutrients such as vitamins C and D, calcium
(Holt, 1999; Slattery et al, 1988), and selenium to name a few.

The non-nutrient components of food and their anti-cancer properties have only
been studied extensively in the past decade or two. Phytochemicals, the naturally
occurring plant constituents, have been linked to reduced cancer risk. Phenolic
phytochemicals are the largest category of phytochemicals and are most widely
distributed in the plant kingdom. Food sources of these compounds range from grains
such as whole wheat, soy and dry beans to vegetables such as onion, kale, and celery and
fruits such as raspberries, apples, cherries and cranberries (King and Young, 1999).
Phenolic compounds in tea and coffee have also been shown to inhibit mutagenesis (Stich
er al., 1982). The anti-carcinogenic properties of these compounds might be linked to

their anti-oxidant and free-radical scavenging properties.

Genetic Basis of Colon Cancer

Cancers differ from other genetic diseases, as they require not one but several mutations
(Vogelstein and Kinzler, 1993). The “multiple hit” hypothesis for the development of
cancer is supported by the relationship between the incidence of most human cancers and
age suggesting that multiple mutations occurring over decades lead to development of
cancer (Knudson, 1985). Colon cancer is understood to be a result of several gerrnline
and somatic genetic mutations. Because the development of colon tumors occurs through
well-defined morphological stages, it has been possible to establish an approximate order
in which these mutations occur (Fearon and Vogelstein, 1990). Tumor suppressor genes

are negative regulators of cell grth and their loss by mutation results in loss of the

crucial “brake” on tumor growth. The initiating mutation in colon cancer is in the tumor
suppressor gene, adenomatous polyposis coli (Apc). The second hit can either be the loss
of the other allele of Apc, or the mutation of one of the other genes implicated in colon
cancer. Mutations in the Apc gene lead to the formation of benign adenomas. Mutation
of the ras gene in one of the benign adenomas can lead to further clonal expansion. This
can be followed by additional mutations in genes such as the p53 or the DCC @eleted in
_C_olon Cancer) gene that can lead to the progression of the benign adenoma to the
malignant carcinoma stage (Fearon and Vogelstein, 1990). A better understanding of
genetic alterations that are involved in the onset and progression of colon cancer has
provided a keener focus on the gene-nutrient interplay in colon cancer and its role in

incidence and progression of the disease.

Colon Cancer and the Age Gene

Mutation of the Apc gene is thought to be one of the earliest mutations according to the
multi-step genetic model for colorectal tumorigenesis proposed by Fearon and Vogelstein
(1990). Germline Apc mutations result in the development of numerous polyps in the
large intestine by the second or third decade of life of patients with FAP (familial
adenomatous polyposis), the inherited, autosomal dominant predisposition to colon
cancer (Kinzler et al., 1991). Apc mutations have been identified in sporadic colorectal
neoplasia as well as in inherited non-polyposis colon cancer, which results from inherited
mutations in DNA mismatch repair gene.

Though loss of normal Apc function is an important step in the early stages of

intestinal tumor formation, it is not clear how this loss contributes to tumor formation.

 

Normal

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Loss of Ape

V

Hyperproliferative
Epithelium
DNA
Hypomethylation
Early Adenoma

K-ras Activation

7

Intermediate

 

 

 

 

 

 

 

 

l Loss of DCC

Late Adenoma

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 Loss of p53
Carcinoma Other
Alteration

 

 

 

 

 

l

Metastasis

 

 

 

 

Figure l. Multistep model for the development of colorectal carcinogenesis
(Modified from Fearon and Vogelstein , 1990)

Recent findings have suggested that APC protein associates with a number of
proteins including the cadherin-binding protein, B—catenin. Wild-type APC has been
shown to inhibit the increased B—catenin/T cell factor (T cf) signalling observed in
colorectal cancers (Munemitsu et al., 1995). The inhibitory activity of APC on [3-
catenin/ch signaling is attributed to its ability to form a macromolecular complex with ,8—
catenin and GSK-BB (Glycogen Synthase Kinase-3fi), thus facilitating the degradation of

B—catenin (Munemitsu er al., 1995).

Age Mutations in Human Colon Cancers

Several hundred somatic and germline mutations have been reported in the Apc gene.
More than 95% of both germline and somatic mutations result in premature truncation of
the polypeptide. Germline mutations are distributed throughout the 5’ half of the gene,
and somatic mutations are concentrated in a mutation cluster region (MCR) (See Fig. 2)
between codons 1286 and 1513. The high frequency of mutations in the MCR in the
more severe cases of polyposis suggests that mutations in the MCR are more effective in
producing tumors than mutations elsewhere in the gene. Mutations occurring before
codon 157 result in a milder form of polyposis marked by fewer polyp numbers and later
stage of onset. Therefore, the severity of the disease, is related to the location of the

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Rodent Models in Colon Cancer Researg!

To study the effects of diet on progression from the initial mutational event to carcinoma
in human subjects is nearly impossible due to the enormous genetic heterogeneity in a
large group of people. In addition, there are difficulties associated with long time control
of diet in these groups. Importance of good animal models to study diet and colon cancer
relationships, therefore, cannot be overemphasized. In experimental work spanning
nearly a century, researchers have tried to reproduce the characteristics of the human
colon cancer in animal models. Most models are based on the use of carcinogenic
chemicals such as dimethylhydrazine or azoxymethane. These chemicals are usually
administered orally or subcutaneously to produce tumors in the intestinal tract.

Recently, the knowledge of genetic changes that accompany the morphological
stages in the process of colon carcinogenesis has led to the introduction of several
transgenic and knock-out mouse models. In these models either one of the tumor
suppressor genes has been mutated (p53 knockout mouse) (Carbone, 1992), or a proto-
oncogene has been activated [mutated M C C (mutated in colon cancer) mouse]. Genetic
mouse models with mutated Apc gene include the Apc 1638 mouse generated by gene
targeting and embryonic stem cell technology, the Apc (5716) mouse, carrying a
truncation mutation at codon 716 of the Apc gene (Pories et al., 1993), and the Min
mouse, which also carries a germline mutation in the Apc gene. A major advantage of
using the mouse models is the ability to experimentally control the genetic background of

the animal.

The Min Mouse

The Apc Min (Multiple Intestinal Neoplasia) mouse developed in the early 90’s (Moser et
al, 1990) carries a germ-line mutation in the Apc gene. N—ethyl-N—nitrosourea (ENU), a
potent germline mutagen of the mouse, was used to induce mutations in the mice to create
mouse models for various human diseases. The Min mouse was discovered upon
phenotypic screening of ENU-treated mice and has become an extremely useful model for
studying human intestinal cancer. The Min mouse carries an autosomal dominant
mutation that has been localized to the region of mouse chromosome 18 that also carries
the Apc gene. Sequence analysis of the Apc cDN A has identified the nonsense mutation
resulting from a T/A -> A/T transversion at nucleotide 2549 of Apc. The mutated gene
codes for a truncated protein product that lacks the binding domains that are crucial for its
function (Figure 2) (Shoemaker et al, 1997). The ability of the APC protein to bind and
regulate fi—catenin, a member of the family of intracellular catenins that regulate cell-cell
adhesion by interacting with E-cadherin is thought to be the key to the involvement of
this protein in tumor formation. It is believed that the truncated protein encoded in the
Min mouse lacks the catenin-binding domains that are essential for regulating fi-catenin
binding to E-cadherin and is unable to regulate the downstream ch-mediated signaling
pathway.

The phenotypic manifestation of the mutation in the Apc gene is the development
of multiple adenomas throughout the intestinal tract. On the C57BIJ6J genetic
background, Min mice develop, on average, 50 or more tumors in the intestinal tract.
Most of the tumors are benign adenomas, probably because the mice die prematurely at

~150 days of age due to secondary effects of tumor growth, such as, severe anemia and

Min Mutation Cluster
Mutation Region

   

 

 

 

. . _ . . . '——1
.1 ,, - . . _; l
|_f_J t | I l . ”I ; J]
homo armadillo Constitutive Kinase-regulated Microtubule
dimerization repeats B—catenin B-catenin association
domain binding domain

Figure 2. Potential Apc functional domains and location of Apc mutations in human
colon cancer and Min mutation. (Adapted from Shoemaker et al., 1997).

intestinal blockage. As described earlier, a majority of human colorectal neoplasia, both
somatic and hereditary, involve loss of one or both alleles of the Apc gene. The Min
mouse somewhat mimics the inherited form of human intestinal cancer, FAP (familial
adenomatous polyposis). Though most of the tumors in FAP patients occur in the colon,

the Min mouse has multiple tumors in the small intestine, and only a few in the colon.

Min Mouse in Nutrition Studies

Following the propagation of the Min mouse model in 1990, it has been used in
numerous studies to study the complex relationship between dietary factors and colon
cancer. Some of the dietary factors studied in the Min mouse include dietary fat, fiber,
calcium, and certain phytochemicals. The results of these studies have been conflicting
and at times appear to contradict the current understanding of the effect of diet in colon
cancer. Diets high in animal and saturated fats are generally associated with high risk of
developing colon cancer and diets rich in vegetables and fruits are associated with lower
risk of colon cancer (Sandler et al., 1993). However in Min mice, intake of high dietary
fat and vegetable-fruit mixture had no effect on the development of intestinal tumors
(Table 1) (van Kraner et al., 1998). A study examining the effect of dietary fiber on
intestinal tumori genesis in the Min mouse showed no protective effects of wheat bran or
resistant starch on small intestinal tumors (Pierre et al., 1997). Short-chain fructo-
oligosaccarides reduced tumors in the colon, but not in the small intestine where the
majority of adenomas occur in Min mice (Pierre et al., 1997). These results contradict
numerous studies that have shown a protective effect of fibers such as wheat bran on

colon cancer risk in clinical

10

Table 1. Studies investigating the effects of dietary factors on tumorigenesis

in the Min mouse

 

 

Study Dietary Component Effect on tumor formation in
ApcMm mouse
Kraner et al, 1998 dietary fat and no effect
fruit- vegetable mixture no effect
Sorensen et al, 1998 soy isoflavones no effect
Paulsen et al, 1997 EPA and DHA enriched fish oil suppressed tumor grth and
formation
Pierre et al, 1997 short-chain fructo- no effect on small intestinal
oligosaccarides tumors, l colon tumors
Kennedy et al, 1996 soybean-derived Bowman-Birk ,1. total tumor number

inhibitor

trials (Macrae, 1999), and carcinogen-induced colon carcinogenesis (T akahashi et al.,
1999; Compher et al., 1999).

However, Kennedy et al (1996) found that feeding diets containing the soybean-
derived Bowman-Birk protease inhibitor reduced tumor number in Min mice when the
dietary treatment was started in utero. These studies suggest that while the effect of
dietary factors is not always what is expected from current hypothesis, beginning the
treatment at an earlier time point may be an important factor.

Paulsen et al. (1997) examined whether the n-3 polyunsaturated fatty acid ethyl
ester enriched fish oil K85 (a mixture of eicosapentaenoic acid and docosahexaenoic acid
as ethyl esters) could inhibit the intestinal tumorigenesis in Min mice. Dietary K85
treatment reduced the number of small intestinal tumors: in males, the maximum
reduction was 66% with 0.4% of K85; and in females, the maximum reduction was 48%
(P = 0.043) with 2.5% of K85, but the inhibition was only slightly increased from 0.4% to
2.5% of K85. The small intestinal tumor diameter was reduced by K85 in a dose-
dependent manner. In the large intestine, the mean number of tumors/mouse was 1.0 +/—
0.5 in males and 0.8 +/- 0.2 in females. Although K85 treatment tended to reduce the
number and diameter of the large intestinal tumors, these effects did not reach statistical
significance.

Sorenson et al. investigated whether soy isoflavones inhibited intestinal tumor
development in Min mice (Sorenson et al., 1998). The mice were fed a Western-type
high-risk diet (high fat, low fiber and calcium) containing two different levels (16 and
475 mg of total isoflavones per kg diet) of isoflavones from soy. No significant

differences in the incidence, multiplicity, size and distribution of intestinal tumours were

12

observed between Min mice fed low and high isoflavone-containing diets. Thus, in
contrast to epidemiological studies, their results demonstrated that high amounts of soy
isoflavones present in a Western—type high risk diet were not protective against intestinal
tumor development in the Min mice. The interventions that have consistently reduced
tumor numbers in Min mice are the Non-Steroidal Anti-Inflammatory Drugs (N SAIDs)
such as aspirin (Barnes and Lee, 1998), sulindac (Beazer-Barclay et al, 1996) and

piroxicam (Jacoby et al, 1996) (Table 1).

Non-Steroidal Anti-Inflammatory Drugs

A growing number of studies suggest that Non Steroidal Anti-Inflammatory Drugs

(N SAIDs) such as aspirin and piroxicam may reduce the risk for colorectal tumorigenesis
(W addell and Loughry, 1983). These drugs have clinical application as the toxicity
associated with their intake is considerably less than with other anti—cancer drugs.

Several epidemiological studies have reported that a daily intake of aspirin can reduce the
risk for death due to colorectal cancer. Sulindac suppressed tumorigenesis in FAP
patients (Labayle et al., 1994).

The NSAIDs, Piroxicam and sulindac, have been shown to significantly inhibit
tumor multiplicity in Min mice. In a study where piroxicam was given to Min mice
continually in the diet at a level of 200ppm beginning at 30 days of age, it reduced tumor
number by a factor of 8 (J acoby et al., 1996). This effect was limited to the small
intestine. The low average colon tumor number (06:03) was cited as the reason why an

effect in the colon was difficult to detect.

l3

In a study by Boolbol et al. (1996), Min mice were given sulindac in drinking
water at 160 ppm at 5-6 weeks of age. After 9 weeks of treatment the tumor number
decreased from 11.9 in the untreated mice to 0.1 in sulindac treated mice. Treatment was
begun after 4 weeks of age, therefore the results represent the effect of these drugs on
tumor promotion and progression rather than initiation. The authors also examined the
intestinal expression of cylcooxygenase-Z (Cox-2) and the levels of enterocyte apoptosis
in untreated and sulindac—treated Min mice to explain the results. They found that tumor-
free intestinal epithelium from Min mice expressed higher level of Cox-2 than the
intestinal epithelium of normal littermates. Williams et al. (1996) also found elevated
levels of Cox-2 mRN A and protein in intestinal tumors from untreated Min mice.
Treatment of Min mice with sulindac reduced Cox-2 to levels observed in normal
littermates. It is not certain at this point if the reduction in Cox-2 expression is
responsible for the reduction in tumor multiplicity or an independent effect. Some studies
suggest a prostaglandin-independent mechanism for the sulindac (Chiu et al., 1997).
Sulindac is a pro-drug that is modified in the liver and by colonic bacteria to its
metabolically active sulfide derivative that possesses the anti-inflammatory activity.

Boolbol et a1 (1996) also reported a reduction in the level of enterocyte apoptosis
in Min mice compared to normal littermates and a reversal of this effect by sulindac.
However, this effect was more pronounced when measured by immunoperoxidase
analysis and not as apparent when measured by terminal tranferase-mediated dUTP nick
end-labeling (TUNEL) assay. Tumor multiplicity in control animals reported in this

study was considerably lower (~12 tumors/animal) compared to the numbers reported by

14

other studies (~50 tumors/animal). This difference might be due to difference in diets fed
or other environmental factors.

Another study reported the effect of sulindac on tumor multiplicity in Min mice
(Beazer-Barclay et al., 1996). High (334 ppm) and low (167 ppm) doses of sulindac in
diet and in drinking water (84 mg/L) were tested. A stronger effect of sulindac was
observed when the treatment was started prenatally by treating pregnant females. This

suggests that sulindac may inhibit tumor formation or induce tumor regression.

Whole Wheat and Cancer
Whole grains such as wheat are important sources of many nutrients including dietary
fiber, resistant starch, oligosaccharides, trace minerals, vitamins and other compounds
such as phytoestrogens and antioxidants (Slavin et al., 1999). Dietary guidelines
recommend the consumption of whole grains to prevent chronic diseases. This view is
supported by epidemiological studies that have shown that consumption of whole grains
such as wheat, rice and corn is protective against cancer, especially gastrointestinal
cancers such as gastric and colon cancer (Block, 1992; Slavin, 1994) and cardiovascular
disease (Brown et al., 1985). In experimental studies, wheat bran was shown to decrease
aberrant crypt foci, preserve normal proliferation and increase apoptosis in
azoxymethane-induced colon cancer (Compher er al., 1999). These anti-neoplastic effects
were associated with increased fecal butyrate levels.

Whole wheat has several components that may protect against colon cancer. It is
a rich source of fermentable carbohydrates including dietary fiber, resistant starch, and

oligosaccharides. Fermentation of these undigested carbohydrates in the colon leads to

15

the production of short-chain fatty acids and gases. Production of short-chain fatty acids
such as butyrate has been linked to a decreased risk of colon cancer. Undigested
carbohydrates also increase fecal weight and decreased intestinal transit time thus limiting
the exposure of intestinal epithelium to mutagenic and tumor-promoting agents such as
bile acids in the fecal matter (W rick et al., 1983).

Whole wheat also contains a number of antioxidants such as vitamins, trace
minerals, and non-nutrients such as phenolic acids, lignans, phytoestrogens and phytic
acid (Thompson, 1994). Whole wheat is a rich source of vitamin E. The vitamins and
minerals are present in the outer layer of the grain and are lost during milling; therefore,
refined flours are a poor source of these nutrients. Whole wheat is also a rich source of
phenolic acids such as ferulic and caffeic acids, which are located in the bran layer.
Durum wheat bran has been shown to have antioxidant properties in an in vitro model
(Onyeneho and Hettiarchchy, 1992). Phenolic acids may also function by inducing
detoxification systems, specifically the phase II conjugation reactions (W attenberg, 1985).
Phytic acid, another constituent of whole wheat, functions as an antioxidant by chelating
various metals thus suppressing damaging iron-catalyzed redox reactions (Graf and
Eaton, 1993). Vitamin E, another antioxidant present in whole wheat at the intracellular
level protecting polyunsaturated fatty acids in the cell membrane from oxidative damage.
Selenium is a trace mineral present in whole wheat that functions as an antioxidant. The
amount of selenium in wheat varies and is proportional to the level of selenium in the soil
in which the crop was grown. Selenium is a cofactor for the glutathione peroxidase

enzyme that protects tissue against oxidative damage. Whole wheat also contains

16

phytoestrogens like lignans and anti—nutritive factors such as phytic acid, which may also

function to reduce cancer risk (Adlercreutz, 1984).

Flax and Cancer
Interest in flaxseed as a component of diet is currently undergoing a moderate resurgence.
Flaxseed contains 35% of its mass as oil, most of which is OI-linolenic acid and linoleic
acid (Ensminger, 1983). Flaxseed is a rich source of omega-3 fatty acids and the richest
source of plant 1i gnans. Lignans are building blocks for lignins, constituents of plant cell
walls and are present in higher plants such as grains, legumes, vegetables, and seeds. The
level of lignans called secoisolariciresinol diglucoside (SDG) present in flaxseed is 100-
800 times higher than the amount present in other plants (Thompson, 1991). Lignans
possess a dibenzyl structure that is altered when they reach the colon. Lignans are
absorbed in the colon. In the liver they are conjugated with glucuronic acid or sulfate,
excreted with bile, deconjugated by the colonic bacteria into mammalian lignans
enterolactone and enterodiol. By virtue of their 2,3-dibenzylbutane structure, these
lignans resemble potent synthetic estrogens, which may act antiestrogenically. Both
enterolactone and enterodiol have been shown to express weak estrogenic and
antiestrogenic properties (Adlercreutz et al., 1986). Antiestrogens have displayed
antitumor activity in experimental and human mammary tumors (Litherland and Jackson,
1988).

Flaxseed lignans have been shown to be inducers of B-glucuronidase activity, the
enzyme responsible for the deconjugation of lignans in the colon (Jenab and Thompson,

1996). B-Glucuronidase, an inducible enzyme is also involved in the enterohepatic

l7

circulation and activation of procarcinogens, carcinogens, mutagens and toxins that may
be associated with an increased risk of colon carcinogenesis (Reddy et al., 1992).
Flaxseed has been shown to have beneficial effects in mammary carcinogenesis in rats
when added to the diet during initiation and promotional stages (Serraino and Thompson,
1992a). In rats treated with azoxymethane, flaxseed meal supplementation of diet at 5%
and 10% reduced the formation of aberrant crypts and aberrant crypt foci, probable

precursors to colonic tumors, in the colon (Serraino and Thompson, 1992b).

Black Beans and Cancer

Anti-carcinogenic effects of black beans were suggested by epidemiological studies
showing a low incidence of colon cancer in many Latin American countries where the
consumption of dry beans is high (Correa, 1981). Dry beans are among the best known
sources of dietary fiber and contain both soluble and insoluble types of dietary fibers
(Hughes et al, 1996, Herrera et al., 1998). In experimental studies, dry beans inhibited
azoxymethane-induced colon carcinogenesis in rats (Hughes et al., 1997). In addition,
beans such as black beans are a rich source of numerous phytochemicals, including
polyphenolics, that possess both anticarcinogenic and antioxidant properties.

The most notable of the phytochemicals present in the black beans is the
anthocyanins. The characteristic red anthocyanin pigment found in the black bean can
potentially be used as a coloring agent in foods. Anthocyanins have been shown to
possess anti-oxidant properties against the potent and highly destructive peroxyl radicals
(Wang et al., 1997). Free radicals and their oxidants cause oxidative damage to lipids,

proteins, and nucleic acids leading to cancer and atherosclerosis. The free radical

l8

neutralizing property of these phytocherrricals, such as those present in the black bean, are

thought to be of central importance to prevention of cancer.

Energy Restriction and Cancer

The inhibitory effect of energy or caloric restriction on cancer has been recognized for
some time. Additionally energy restriction is perhaps the only dietary treatment shown to
improve longevity in laboratory animals. The association between energy restriction and
cancer protection was established by work done almost a century ago. In 1909, Moreschi
(Moreschi, 1909) was the first to report the effects of underfeeding on carcinogenesis. He
observed that sarcoma transplanted into mice grew more slowly as less and less food was
offered. In 1914, work published by Rous (Rous, 1914) showed that underfeeding also
inhibited the growth of spontaneous tumors. By 1943, Tannenbaum (T annenbaum, 1943)
had shown that energy restriction inhibited the growth of both spontaneous and induced
tumors. Energy restriction was shown to reduce colon tumor growth in the rats treated
with azoxymethane (Pollard et al, 1984; Reddy et al, 1987). This cancer inhibitory effect
of caloric restriction seems to be independent of the effect of fat in the diet. In chemically
induced models of colon and mammary cancers, carcinogenesis was inhibited in 40%
calorie-restricted rats even as they were fed increased percentage of dietary fat (Klurfeld
et al, 1987). In rats, energy restriction at middle age (16 weeks) modulated the
development of pre-neoplastic lesions (Lasko, 1999). Energy restriction decreased the
total number of aberrant crypt foci, the precursors to colonic tumors.

Despite the abundance of literature on energy restriction and inhibition of cancer,

little is known about the mechanisms underlying anti-neoplastic effects of energy

19

restriction. One possible mechanism suggests an effect of energy restriction on the
hormonal balance in the animal. Energy restriction has been shown to stimulate the
pituitary-adrenocorticotropic axis, which results in decreased levels of mitogenic and
reproductive hormones (Ames and Shigenaga, 1994; Leakey et al, 1994; Weindruch and
Walford, 1988). This energy restriction-induced switch from a reproductive pattern of
life to one emphasizing repair and maintenance functions has been suggested to reduce
tumor incidence by lowering the metabolic rate, hence reducing the production of toxic
metabolic by—products and reducing nuclear damage. Energy restriction reduces cell
proliferation and increases cell death by apoptosis in animals (W achsman, 1996). Energy
restriction was shown to reduce colon weight, the total number of cells, DNA synthesis
and the total number of crypts. The reduction in the total number of cells dividing at any
given time was suggested to impart resistance against induction of colon cancer to these
animals (Albanes et al., 1990). Up—regulation of apoptosis has also been cited as a
possible mechanism by which energy restriction imparts its anti-carcinogenic effect

(W achsman, 1996). A chronic dietary restriction was shown to increase the spontaneous
apoptotic rate and decrease cell proliferation rate in hepatocytes of 12-month old B6C3F1
mice (James et al., 1998). Inhibition of cell proliferation and increased cell death by
apoptosis may account for the anti-cancer effect of energy restriction.

Cellular proliferation and death are regulated by the expression of transcription
factors that regulate cell cycling. Zhu et al. (1999) have studied the effects of energy
restriction on expression of cell-cycle protein in chemically induced mammary
carcinogenesis. Energy restriction, in a dose dependent manner, up-regulated p27/kip1, a

gene product associated with cell-cycle growth arrest, and down-regulated cyclin D1, a

20

protein that combines with cyclin-dependent kinases to promote phosphorylation of
retinoblastoma protein and the progression of cell through the cell-cycle (Zhu et al.,
1999). Another recent publication reported the changes in gene expression in response to
aging and energy restriction (Lee et al, 1999). Using a novel technique called
oligonucleotide based microarrays, researchers examined the expression of over 6,000
genes, as reflected by mRN A levels. The study showed that several changes in
expression of genes as a result of aging, were partially reversed by energy restriction. The
genes involved in energy metabolism that were down—regulated in aging animals were up
regulated in energy restricted animals. Similarly, the genes involved in protein
metabolism, and biosynthesis were also up-regulated by energy restriction. In contrast,
the genes involved in stress response, e. g., the heat shock response, DNA—damage-
inducible genes, and the oxidative genes that were up-regulated in aging animals, were
down-regulated in energy restricted animals (Lee et al, 1999). These studies provide
evidence for the hypothesis that energy restriction elicits its effects by modulating gene

expression.

Complementag DNA Arrays

Oligonuceotide based microarrays or cDN A microarrays have been in use for a short time
(Eggers er al, 1994; Fodor er al, 1991; Lamture et al, 1994), but their use is increasing in
the medical research community. Microarray technology is a powerful tool for functional
genomics research. Complimentary DNA arrays enable the simultaneous detection of
thousands of genes in a single small sample. The microarrays consist of large numbers of

cDNA molecules spotted in a systematic order on a solid substrate (such as a nylon

21

membrane, glass slide or silicon chip). DNA arrays are fabricated by high-speed robotics
on glass or nylon substrates, for which labeled probes are used to determine
complementary binding allowing parallel gene expression and gene discovery studies.
Oligonucleotide microarrays are fabricated either by in situ light-directed combinational
synthesis or by conventional synthesis followed by immobilization on glass substrates.
Sample DNA is amplified by the polymerase chain reaction (PCR) and a fluorescent label
is inserted and hybridized to the microarray. This technology has been successfully
applied to the simultaneous expression of many thousands of genes and to large-scale
gene discovery, as well as polymorphism screening and mapping of genomic DNA clones
(Ramsay, 1998). The excitement surrounding microarray technology has been tempered
by the limited ability of the general biomedical research community to gain access to it.
Only recently, the hardware required for exploitation of the technology has become
increasingly available (Bowtell, 1999).

Expression of selected genes coding for proteins with defined cellular functions
has been analyzed in human cell lines derived from normal colonic mucosa, non-
mucinous colonic carcinomas and mucinous colonic carcinomas (Backert et al., 1999).
The expression of 10 genes was found to be altered in colon carcinoma cells. The
topoisomerase H alpha and the mitosis inhibitor WEElHu gene were significantly
suppressed in the tumor cell lines. In addition, the gene coding for the cell cycle inhibitor
p21 was overexpressed only in cell lines derived from mucinous carcinomas. These
alterations were confirmed by northern blotting or semi-quantitative reverse transcription-

polymerase chain reaction (RT-PCR) suggesting that the cDNA array method permits a

22

correct identification of changes in gene expression with a relatively high accuracy
(Backert etal., 1999).

The development and use of molecular-based therapy for breast cancer and other
human malignancies will require a detailed molecular genetic analysis of patient tissues.
In a recent study, the laser capture microdissection and high density cDNA arrays has led
to the generation of gene expression profiles of cells from various stages of tumor
progression as it occurs in the actual neoplastic tissue milieu. Complimentary DNA
microarrays were used to monitor in vivo gene expression levels in purified normal,
invasive, and metastatic breast cell populations from a single patient. The use of cDNA
microarray analysis combined with laser capture microdissection has provided a powerful
new approach to elucidate the in vivo molecular events surrounding the development and
progression of breast cancer and is generally applicable to the study of malignancy (Sgroi,
1999).

The microarray analysis of gene expression profiles has been used to characterize
and distinguish the mechanisms of response of colonic epithelial cells to physiological
and pharmacological inducers of cell maturation. The short—chain fatty acid butyrate,
produced by microbial fermentation of dietary fiber in the large intestine, is a
physiological regulator of major pathways of colonic epithelial cell maturation: cell cycle
arrest, lineage-specific differentiation, and apoptosis. Microarray analysis of 8,063
sequences of SW620 colonic epithelial cells upon treatment with butyrate was conducted
and compared with the effects of sulindac, the nonsteroidal anti-inflammatory drug with
significant chemopreventive activity for colon cancer, and curcumin. Curcumin, a

component of mustard and curry is structurally and functionally related to sulindac that

23

also has chemopreventive activity. Although butyrate and sulindac induce a similar G0-
G1 arrest, decrease in B-catenin-ch-LEF signaling, and apoptotic cascade, there were
striking differences in the gene clusters effected by these compounds. This has important
implications for characterization of chemopreventive agents and recognition of potential
toxicity and synergies (Mariadason, 2000).

This potential of cDNA microarray technology in the identification of carcinogens
and other environmental hazards and in determination of the relative safety of natural or
synthetic chemicals to which humans are exposed is great. The use of this methodology
will be critical to increase the sensitivity of detection of the potential toxic effects of
environmental chemicals and to understand their risks to humans (Afshari et al., 1999).

In the past years, microarray technologies have moved beyond the proof-of-
principle stage and are now being used for genome-wide expression monitoring, large-
scale polymorphism screening and mapping, and for the evaluation of drug candidates
(Braxton, 1998). Large-scale RNA assays and gene-expression-microarray studies are
being applied at several stages in the drug-development process and could ultimately have
broad applications in disease diagnosis and patient prognosis (Zweiger, 1999).

Microarray analysis has been exploited to understand the function of genes such
as MYC and BRCA-l (Coller er al., 2000). MYC affects normal and neoplastic cell
proliferation by altering gene expression, but the precise pathways have always remained
unclear. Oligonucleotide microarrays were used to analyze expression of 6,416 genes to
determine changes in gene expression caused by activation of c-MYC in primary human
fibroblasts. In these experiments, 27 genes were consistently induced, and 9 genes were

repressed. The identity of the genes revealed that MYC may affect many aspects of cell

24

physiology altered in transformed cells: cell growth, cell cycle, adhesion, and cytoskeletal
organization. Identified targets included the nucleolar proteins nucleolin and fibrillarin,
the eukaryotic initiation factor 5A, G1 cyclin D2 and the cyclin—dependent kinase binding
protein CksHsZ, cyclin-dependent kinase inhibitor p21 (Cipl), the extracellular matrix
proteins fibronectin and collagen, the cytoskeletal protein tropomyosin, and tumor
necrosis factor receptor associated protein TRAPl (Coller er al., 2000).

The breast and ovarian cancer susceptibility gene product BRCAl has been
reported to play an integral role in certain types of DNA repair. High density cDN A array
screening of colon, lung, and breast cancer cells identified several genes affected by
BRCAl expression in a p53-independent manner, including DNA damage response genes
and genes involved in cell cycle control. Notable changes included induction of the
GADD45 and GADD153 genes and a reduction in cyclin B1 expression. Therefore,
BRCAl has the potential to modulate the expression of genes and function of proteins
involved in cell cycle control and DNA damage response pathways (MacLachlan, 2000).

Microarrays are one of the latest breakthroughs in experimental molecular
biology, which allow monitoring of gene expression for tens of thousands of genes in
parallel and are already producing huge amounts of valuable data. Analysis and handling
of such data are becoming major bottlenecks in the utilization of the technology (Brazma
and Vilo, 2000). This shift in the biological investigation paradigm, such that the
bottleneck in research is shifting from data generation to data analysis, has led to the use
of hierarchical clustering, divisive clustering, self-organizing maps and k-means
clustering to make sense of this mass of data (Sherlock, 2000). Microarray-based studies

are uncovering broad patterns of genetic activity, providing new understanding of gene

25

functions and, in some cases, generating unexpected insight into transcriptional processes
and biological mechanisms. One topic that has come to the forefront is how best to
effectively manage and interpret the large data sets being generated. Although progress
has been made, this remains a challenging opportunity for functional genomics research
(Harrington, 2000).

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32

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33

CHAPTER II

INHIBITION OF INTESTINAL TUMORIGENESIS IN APCMIN MICE BY FOOD

RESTRICTION, DEF ATTED FLAX/WHOLE WHEAT AND BLACK BEANS

Mridvika, M.R. Bennink, and L.D. Bourquin

Department of Food Science and Human Nutrition, Michigan State University,
East Lansing, MI 48824-1224

34

ABSTRACT

ApcMin (or Min) mice develop multiple adenomas in the small intestine (SI) and
have been used to study the effects of diet on intestinal cancer with mixed results. We
examined the effects of 20% food restriction, feeding a defatted flax/whole-wheat flour
based diet and feeding a black bean-based diet for 11 weeks on tumor number and size in
small intestine and colon. The NSAID drug sulindac was used as the positive control.
Results: SI tumor number decreased (P<0.05) from 765:5.8b in the casein fed control
group to 45.4i5.3a in the sulindac group (42% decrease), 53.2155" in the flax/wheat
group (30% decrease), 55.6:57a in the black bean group (28% decrease) and 48.9_-I;9.9al in
the diet restricted group (36% decrease). Sulindac reduced average SI tumor diameter
from 1.32 to 1.03 mm (P<0.01). Black bean and flax/wheat diets increased SI tumor
diameter (P<0.05). Colon tumor numbers did not differ significantly among groups.
Black bean and flax/wheat diets increased the colon tumor volumes compared to the
control group. Conclusion: black bean, flax/wheat diets and 20% dietary restriction
reduced the number of adenomas in SI. However black bean and flax/wheat diets

increased 81 and colon tumor size. These treatments did not reduce colon tumor number.

35

INTRODUCTION

The ApcMin (or Min: Multiple Intestinal Neoplasia) mouse is a model for human
familial adenomatous polyposis (FAP), a hereditary condition in humans that progresses
to colon cancer early in life if left untreated. The genetic defect is a nonsense mutation of
the Apc gene, a gene implicated in both sporadic and inherited (e.g. FAP) human
colorectal cancers (Powell et al., 1992; Kinzler and Vogelstein, 1996). More than 95% of
both germline and somatic mutations of the Apc gene result in premature truncation of the
polypeptide. Functional APC has been shown to associate with more than half a dozen
proteins including [i-catenin and glycogen synthase kinase-313 (Su et al., 1993). Wild-
type APC has been shown to inhibit the increased B—catenin/T-cell factor (T cf) signalling
observed in colorectal cancers (Munemitsu et al., 1995). The inhibitory activity of APC
on B-catenin/ch signaling may be due to its ability to form a macromolecular complex
with B-catenin and GSK-3fi, thus facilitating the degradation of B—catenin (Rubinfeld et
al., 1996). The truncated APC protein in Min mice lacks the binding domains necessary
to form the multimolecular complex with B—catenin and GSK—3/3. Because of its central
role in regulating B—catenin signalling, APC is often referred to as a gatekeeper protein.
In the absence of this vital gatekeeper function, as occurs in the Min mouse, regulatory
controls of the cell cycle are disabled leading to increased cell proliferation, delayed
differentiation and aCCun'rulation of additional mutations. This ultimately results in the
formation of multiple tumors in the colons of FAP patients and multiple adenomas
throughout the intestinal tract in Min mice.

Following the introduction of the Min mouse strain (Moser et al., 1990), several

researchers examined the relationship between tumor development in Min mice and

36

dietary fat, fiber, calcium, and certain phytochemicals. Results of these studies have been
inconsistent. For instance, it is generally believed that high intakes of animal and
saturated fat are associated with a high risk of developing colon cancer. Conversely, diets
rich in vegetables and fruits are associated with a reduced risk of colon cancer (Sandler et
al., 1993). However, a study examining the effects of dietary fat and a vegetable-fruit
mixture on adenoma formation in Min mice (van Kraner er al., 1998) found no effect of
dietary fat (40% vs 20%) or the vegetable-fruit mixture on the development of intestinal
tumors. Another study examining the effect of dietary fiber on intestinal tumorigenesis in
the Min mouse showed no protective effects of wheat bran or resistant starch on small
intestinal tumors (Pierre et al., 1997). These results contradict numerous studies that
have shown a protective effect of fibers such as wheat bran on colon cancer risk in
clinical trials (Marcae, 1999) and carcinogen-induced colon carcinogenesis (T akahashi et
al., 1999; Compher et al., 1999). Short-chain fructo-oligosaccarides reduced tumors in
the colon, but not in the small intestine where the majority of adenomas occur in Min
mice (Pierre et al., 1997). Kennedy et al. (1996) observed a significant reduction in tumor
number in the small intestine of Min mice fed diets containing the soybean-derived
Bowman-Birk protease inhibitor. The agents that have consistently reduced tumor
numbers in Min mice are the Non Steroidal Anti Inflammatory Drugs (N SAIDs) such as
aspirin, sulindac and piroxicam (Barnes and Lee, 1998; Beazer-Barclay et al., 1996;
Jacoby et al., 1996).

Energy restriction inhibits the growth of both spontaneous and induced tumors in
the liver (T annenbaum, 1940; Tannenbaum, 1943) and reduces colon tumor growth in

rats treated with azoxymethane (Reddy et al., 1987). The inhibitory effect of energy

37

restriction on cancer development is independent of the effect of dietary fat, as
chemically-induced colon and mammary cancers were inhibited by 40% energy-
restriction in rats even as they were fed diets with a higher percentage of fat (Kritchevsky
et al., 1984; Klurfeld et al., 1987). Anti-carcinogenic effects of black beans were
suggested by epidemiological studies showing a low incidence of colon cancer in many
Latin American countries where the consumption of dry beans is high (Correa, 1981).
Beans such as black beans are a rich source of numerous phytochemicals, including
polyphenolics, that possess both anticarcinogenic and antioxidant properties (T akeoka et
al., 1997; Griffiths, 1981). In addition, dry beans are among the best known sources of
dietary fiber and contain both soluble and insoluble types of dietary fibers (Hughes et al.,
1996; Herrera er al., 1998). In experimental studies, dry beans inhibited azoxymethane-
induced colon carcinogenesis in rats (Hughes et al., 1997). Flaxseed, a rich source of
mammalian lignan precursors such as secoisolariciresinol diglycoside, has been shown to
have protective effects in mammary (Serraino and Thompson, 1992a) and colon
carcinogenesis (Serraino and Thompson, 1992b; Jenab and Thompson, 1996). Colonic
bacteria convert plant lignans, such as those found in flaxseed, into mammalian lignans,
mainly enterolactone and enterodiol. These compounds have been suggested to possess
anti-proliferative activity (Herano et al., 1990). Whole-wheat flour was added to the
defatted flax diet to improve the amino acid score of this diet. Wheat also contains
phytochemicals with antioxidant properties such as phytic acid and phenolic compounds
(Slavin et al., 1999).

The present study examined the effect of feeding four diets on tumor number and

size in the small intestine and colon in the Min mouse. The dietary treatments were: food

38

restriction (FR), black bean (Phaeolus vulgaris) diet (BB), defatted flax/whole-wheat
flour diet (FW), a control group (casein diet) (CT) and positive control group that
received 200 ppm sulindac in drinking water (SL). These diets have been shown in
epidemiological studies and initiation-promoter models to have a protective role in colon
cancer. Our objective was to determine the anti-neoplastic effects of these dietary factors

in Min mice.

39

MATERIALS AND METHODS
Exmrimental Design- Min mice were obtained from a breeding colony maintained on
site by crossing C57Bl/6J Min male carrier mice with normal C57Bl/6J females. Mice
were not genotyped before assignment to treatment. Based on 50% incidence of carriers
in progeny and target of 25 Min mice per treatment group, a total of 50 mice (carriers and
non-carriers) were assigned to each treatment group. The actual number of Min mice for
each treatment was between 21 and 26. Distribution of males and females between
treatments was kept as even as possible, except the food restriction group had 20 males
and 2 females. Mice were weaned at 4 weeks of age, assigned to dietary treatments in the
fifth week and randomly assigned to one of these treatment groups: control (CT), food
restriction (FR), black bean (BB), defatted flax/whole-wheat (FW) or sulindac (SL)
group. Mice were weighed weekly. The dietary treatment period was 11 weeks, after
which mice were killed by carbon-dioxide asphyxiation and the number of tumors in

small intestines and colons were counted.

Animal housing- Mice were housed in a temperature (70-72 F) and humidity (40-70%)
controlled room in plastic cages in accordance with NIH, USDA and university
guidelines for laboratory animal research. All mice were housed in plastic cages. Mice in
the FR treatment group were housed individually. All other mice were housed 2-4

mice/box with same sex littermates.

Animal diets- All treatment groups were fed powdered diets that were similar in macro-

nutrient composition (approximately 16.7% fat, 18.9% protein, 20% fiber). The control

40

Table 1. Composition of the control, black bean and defatted flax/whole-wheat diets.

 

 

Ingredient Control Black Bean Defatted F 101/
Whole-Wheat
gfl<g g/kg g/kg
Casein 222.2 - -
Black bean flour - 711.1 -
Wheat flour - - 350
Defatted flax flour - - 400
Sucrose 355 - 51.9
Soybean oil 166.7 156.0 142.3
Solka-floc fiber 200 76.8 -
Mineral mix (AIN-93G-MX) 38.89 38.89 38.89
Vitamin mix (AIN-93-VX) 11.11 11.11 11.11
L-Cysteine 3.33 3.33 3.33
Choline bitartarate 2.78 2.78 2.78
Tert-butylhydroquinone 0.02 0.02 0.02

 

 

41

diet was a modified AIN 93-G diet with a higher fat (16.7%) and fiber (20%) content than
the standard AIN-93G diet (Table 1). Diet ingredients were purchased from Dyets Inc.
(Bethlehem, PA). Defatted flax flour and whole-wheat flour were combined to provide
all of the essential amino acids for mice. All diets except the FW diet had additional fiber
(Solka floc) added to provide the same fiber content as the FW diet. To obtain the black
bean flour, the beans were soaked overnight, cooked at 90-95 °C until soft, dried at 50-55
°C and ground to a meal. Sulindac (Sigma Chemical Company, St. Louis, MO) was
added to the drinking water (200 mg/L; pH 7) of mice in the positive control group (SL).
For the FR treatment group, the amount of the control diet consumed by mice was
estimated in a pilot study where mice were housed in wire bottom cages and food
disappearance and food spillage were measured. Based on these results, the FR male and
female mice were fed 3.0 and 2.5 grams of control diet each day, which corresponded to

approximately 80% of ad libitum consumption.

Tissue Preparation- The small intestines and colons were opened longitudinally,
emptied of their contents, and rinsed with water. The ceca were not fixed or examined for
this experiment. The colons were pinned flat and the small intestines were placed on a
sheet of filter paper and pressed flat with a glass rod. A second sheet of filter paper was
placed over the flattened small intestines. Both small intestines and colons were fixed
overnight in 10% neutral buffered formalin. The tissues were stained in a solution of
0.2% methylene blue in water and stored in 1% formalin in phosphate buffered saline
(PBS) until neoplasms were counted. Tumor number and tumor diameter in the small

intestine were measured under a dissecting microscope at 15x magnification. In the

42

colon, only raised tumors were counted and three-dimensional diameters measured. An

average tumor diameter was computed to calculate tumor volume, (it x dia3)/6.

Statistical Analysis- The data were analyzed by two-way analysis of variance (treatment
x sex). When there was a significant treatment effect (P<0.05), multiple comparisons
were conducted using the Least Significant Difference method. The colon tumor number
data was analyzed with Wilcoxon’s Rank Sum Test as the data were not normally

distributed.

43

RESULTS
Three female and 1 male mice in the BB group died during the 11 week dietary
treatment period. The tumor data from these animals were not collected and hence is not

included in the results.

M The maximum weight gains over the 11-week study period are reported in
Figure 1. Maximum weight gain was defined as: maximum weight during the study
period - initial weight at the beginning of the study. The difference in weight gain
between males and females was not statistically significant. As expected, the weight gain
by the FR group, fed approximately 20% less food compared to the CT group, was
significantly lower than the CT group. The weight gains for animals in BB, FW and SL

groups were not significantly different from animals in the CT group.

Small Intestinal Adenomas There was a strong trend for female mice to have more
adenomas than male mice (P<0.0545)(Table 2). There was a trend for female mice to
have smaller adenomas than males (Table 2). Mice in the CT group had an average of
76.5 :t 5.8 small intestinal adenomas (Table 3). The SL group (positive control) had 42%
fewer adenomas than the CT group whereas treatments FW, BB and FR reduced small
intestine tumor number by approximately 30%, 28% and 36%, respectively. These
reductions in small intestinal tumors were highly significant (P<0.005). Average
adenoma diameter observed in the SL group was significantly smaller than that found in
the CT group, whereas adenoma diameters were significantly larger in the FW and BB

groups compared to the CT group. Small intestinal tumor diameter was unchanged by FR.

44

 

 

l

l

1

l

 

oemoorstnoulxioo
I
Z

 

1

 

 

 

 

Figure 1. Weight gain in mice in treatment groups control diet (CT), defatted flax/ whole
wheat diet (FW), sulindac (SL), black bean based diet (BB) and feed restricted
diet (FR). The weight gain in FR group was significantly lower than the CT
group (P<0.05). The weight gains in other four groups were statistically
similar.

45

Table 2. Overall gender effects in SI and colon adenoma
numbers and sizes in min micel.

 

Female Male P Value
(n=49) (n=63)
SI Adenoma Number 61.7 (4.9) 50.1 (3.5) 0.0545
SI Adenoma Diameter (mm) 1.41 (0.04) 1.49 (0.03) 0.0804
Colon Adenoma Number 1.2 (0.33) 1.4 (0.23) 0.6020
Colon Adenoma Volume 3.19 (1.08) 3.93 (0.77) 0.5727
(mm3)

 

 

Data are expressed as least square means (standard error of the means). Differences
in carcinogenic parameters between male and female mice were analyzed with two
way analysis of variance.

46

Table 3. Small intestinal tumor number and diameter in Min mice fed the
control diet and treatment diets].

 

 

Treatment n SI tumor number SI Tumor size
Control 21 76.5 (5.8)b 1.32 (0.04)“
Sulindac 26 45.4 (5.3)a 1.03 (0.04)a
Black bean 22 55.6 (5.7)a 2.08 (0.04)(1
Defatted flax 24 53.2 (5.5)8 1.65 (0.04)c
Food restricted 22 48.9 (9.9)‘51 1.22 (0.04)b

 

 

1Data are expressed as least square means (SEM). Differences between treatments were
analyzed by two-way analysis of variance followed by Least Significant Difference test
for multiple comparisons. Means in a column with different superscripts denote

significant differences (P<0.05).

47

Colon Adenomas The number of adenomas was not statistically different between male
and female mice (Table 2). The number of colonic adenomas ranged from 0-3 per mouse
and there was no significant treatment effect on colon tumor number (P>0.05). (Table 4).
The adenomas in the FW-fed group were significantly larger compared to the SL group
(p<0.05) and the adenomas in the BB group were larger than in the SL and CT groups

(p<0.05).

48

Table 4. Colon tumor number and volume in Min mice fed the control and
treatment diets.

 

 

Treatment n Colon tumor number Colon Tumor volume
(m3)

Control 21 1.0 (0.4) 2.74 (1.29)ab

Sulindac 26 2.1 (0.3) 0.48 (1.16)a

Black bean 22 1.2 (0.4) 7.20 (1.27)°

Defatted flax 24 1.1 (0.4) 5.69 (1.22)bc

Food restricted 22 1.2 (0.4) 1.69 (2.19)a

 

 

Data are expressed as least square means (SEM). There were no significant differences

in colon tumor numbers (W ilcoxon’s Rank Sum test, P<0.05). Colon tumor volume

were calculated using the formula v=(rt x diameter3 )/ 6. The colon tumor volumes

were analyzed using one-way analysis of variance followed by Least Significant

Difference test. Means in a column with different superscripts denote significant

differences (P<0.05).

49

DISCUSSION

An inverse relationship between the consumption of NSAIDs and intestinal cancer has
been observed in both human epidemiological studies and animal tumor models. In
humans, regular use of N SAIDS has been associated with a 40-50% decrease in colon
cancer incidence (Thun et al., 1991; Giovanucci et al., 1995). Administration of 334 ppm
sulindac in drinking water decreased intestinal tumor number in Min mice by 38%
(Beazer-Barclay et al., 1996). The 38% reduction in tumor number reported by Beazer-
Barclay et al. is in agreement with the 42% reduction we observed. Some researchers
have reported a much higher level of tumor reduction (90%) with sulindac (Boolbol et al.,
1996). The observations that Min mice have elevated levels of cyclooxygenase-2 (Cox-
2), an enzyme involved in prostaglandin (PG) metabolism, and a decreased fraction of
enterocytes undergoing apoptosis may explain tumor inhibition by N SAIDs. Sulindac has
been shown to block the overexpression of Cox-2 and to restore apoptosis in Min mice
(Boolbol et al., 1996). However, inhibition of PG biosynthesis is not required to observe
an antitumor effect with sulindac. Piazza et al. (1995) have shown the inhibition of
azoxymethane-induced colon carcinogenesis with sulindac sulfone, a metabolite of
sulindac that lacks Cox inhibitory activity. In Min mice, regression of pre-existing
tumors has also been shown to be independent of prostaglandin biosynthesis (Chiu et al.,
1997). Both sulindac sulfone and sulfide have been shown to induce apoptosis in HT-29
human colon carcinoma cells, suggesting that induction of apoptosis might be responsible
for anti-neoplastic effect of sulindac (Piazza et al., 1997).

The 28% reduction in small intestine tumor number by the black bean diet is

consistent with epidemiological data (Correa, 1981) and a rat tumor study (Hughes et al.,

50

1997). Persons in Latin American countries, where dry beans such as black beans are a
staple, have a low incidence of colon cancer (Correa, 1981). Hughes et al. (1997)
observed an inhibition of azoxymethane-induced colon cancer in rats fed pinto beans.
The effect of feeding dry beans on adenoma formation in Min mice has not previously
been reported. Black beans contain a number of factors that have traditionally been
studied by nutritionists because they interfere with protein digestibility and availability
and trace mineral bioavailability. Current research suggests that these anti-nutritive
factors may have anti-cancer properties. Certain foods, especially colorful fruits and
vegetables, contain a variety of compounds such as flavones, flavanones, anthocyanins
and catechins that may offer antioxidant protection against free radicals that can cause
oxidative damage which may be an important factor in the development of cancer (Ames
and Shigenaga, 1994). Black beans are a rich source of the intense red anthocyanin
pigments (conc. 213:2 mg/ 100 g bean), which occur primarily in the skin of the black
beans (Takeoka et al., 1997). The potent antioxidant properties of anthocyanins against
the peroxyl radical have been confirmed (Wang et al., 1997). Anthocyanins from tart
cherries have been shown to inhibit-adenoma formation in Min mice (Kang et al., 2000).
Black beans also contain polyphenols such as tannins (Griffiths, 1981) and tannins have
been shown to possess anticarcinogenic properties in several in vitro studies. Despite a
decrease in SI adenoma number in response to BB diet in our study, the largest SI and
colon tumors were seen in the BB group. We observed that mice with large tumors in the
small intestine had extensive bleeding from the centers of the tumors. The large

adenomas might explain the death of four mice in the BB treatment group.

51

Flaxseed has been shown to possess anti—tumorigenic properties in carcinogen-
induced mammary tumors (Thompson et al., 1996). Rats fed a diet containing 2.5%
defatted flax flour had a significant reduction in the number of aberrant crypt foci in the
colon (Jenab and Thompon, 1996). Defatted flaxseed is a rich source of mammalian
lignan precursors such as secoisolariciresinol diglycoside (Thompson et al., 1991) that
can yield mammalian lignans (enterodiol and entrolactone) by the action of the colonic
bacteria. These molecules closely resemble synthetic estrogenic compounds and their
anti-tumor activity may be due to estrogenic/antiestrogenic effects. Additionally, the
lignan secoisolariciresinol diglycoside has been shown to have hydroxyl radical-
scavenging properties in an in vitro system (Prasad, 1997). The reduction in tumor
number in the small intestine in the FW group may also be due to the presence of
antioxidant phytochemicals in whole-wheat flour. Wheat is a source of phenolic acids,
which are primarily concentrated in the bran fraction of the wheat. Phenolic acids present
in wheat have been shown to possess antioxidant properties in an in vitro assay
(Oneyeneho and Hettiarachy, 1992). In our experiment, feeding the FW diet led to a
reduction in the number of small intestinal adenomas formed but the diet also resulted in
a significant increase in the small intestinal adenoma size. This suggests that eating
defatted flax and wheat flour inhibited the formation of adenomas but enhanced the
growth of adenomas once the adenoma became established. The precise mechanism for
this effect is not clear, however, these diets may contain hitherto unrecognized pro-
angiogenic factors that may facilitate tumor growth.

Energy restriction has been shown to inhibit the growth of spontaneous tumors

(Rous, 1914) and induced tumors (Tannenbaum, 1943). Energy restriction was also

52

shown to reduce colon tumor growth in rats treated with azoxymethane (Pollard et al.,
1984; Reddy et al., 1987). This cancer inhibitory effect of caloric restriction appears to
be independent of the effect of fat in the diet. In chemically induced models of colon and
mammary cancers, carcinogenesis was inhibited in 40% calorie-restricted rats even
though they were fed diets with higher percentages of fat compared to ad libitum fed
control animls (Klurfeld et al., 1987).

The mechanisms underlying the effects of energy restriction are poorly
understood. Food restriction has been shown to stimulate the pituitary-
adrenocorticotropic axis, which results in decreased levels of mitogenic and reproductive
hormones (Leakey et al., 1994;Weindruch and Walford, 1988). This energy restriction-
induced switch from a reproductive pattern of life to one emphasizing repair and
maintenance has been suggested to reduce tumor incidence by reducing the production of
toxic metabolic by-products and reducing nuclear damage. Furthermore there is evidence
that cell proliferation is decreased (Albanes, 1990) and cell death by apoptosis is
increased in energy restricted animals (W achman, 1996; James et al., 1998). This energy
restriction-induced increase in cell death by apoptosis coupled with a decrease in
proliferation could explain the reduction in adenoma formation observed in our study.

Energy restriction can alter gene expression. Zhu et al. (1999) reported that
energy restriction, in a dose dependent manner, up-regulated p27/kip1, a protein
associated with cell cycle growth arrest, and down-regulated cyclin D1, a protein that
promotes the progression of cells through the cell cycle implying that energy restriction
may inhibit cell proliferation. In addition, Lee et al. (1999) have shown the energy

restriction-induced change in gene expression of hundreds of genes involved in aging.

53

Their study showed that changes in gene expression as a result of aging, particularly the
genes involved in energy metabolism, protein metabolism, biosynthesis, and stress
response, were partially reversed by energy restriction (Lee et al., 1999). These studies
provide evidence that energy restriction elicits its effects by modulating gene expression.
Energy restriction may inhibit tumor promotion or progression by modulating expression
of oncogenes or tumor suppressor genes. In the Min mouse, which carries one mutated
Apc allele, food restriction significantly reduced the formation and growth of adenomas,
suggesting that food restriction modulates promotion and progression of carcinogenesis.
As a heterozygous Apc mutation by itself is not enough for the formation of adenomas
and since further mutations must occur for tumors to form, energy restriction must
influence the occurrence of these subsequent mutational events.

Previous to the present study, the Min mouse model had been used in colon
cancer-diet studies with mixed results. In this study, we clearly demonstrate a significant
effect of diet on adenoma formation in the small intestine in mice heterozygous for
mutated Apc gene. Feeding a defatted flax/ whole-wheat flour, a black bean flour-based
diet, or a 20% diet restriction reduced the number of adenomas in the small intestine of
Min mice by approximately 28%. The colon tumor number was not influenced by the
dietary treatments tested in this study. This could be due to the very low number of
adenomas formed in the colon. The shortened life span of these mice due to small
intestinal bleeding might be responsible for the low numbers of colon adenomas seen in
this model. Colon tumorigenesis is probably affected by factors not operative in the small
intestine such as fermentation products in fiber-rich diets. These potential effects were

probably not detected in this study due to model limitations.

54

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59

CHAPTER IH

ENERGY RESTRICTION INHIBITS TUMORIGENESIS AND ALTERS GENE

EXPRESSION IN APCMIN MICE.

Mridvika', M.R. Bennink], M. Fieldenz, and T. Zacharewskiz

Departments of Food Science and Human Nutrition1 and
Biochemistry and Molecular Biologyz,
Michigan State University, East Lansing, MI.

60

ABSTRACT

The ApcMin (+/-) (Min) mice carry a germ-line mutation in the APC gene, and
develop multiple adenomas in the small intestine early in life. Inhibition of intestinal
tumorigenesis in Min mice after 11 weeks of 20% food restriction has been shown
previously (Mridvika and Bennink, 2000). The objectives of this study were: 1) to
determine effects of feeding 30% energy restricted (30% fewer calories from
carbohydrates) vs. control diet (modified AIN-93) for 3, 6 and 10 weeks on adenoma
growth and formation in Min mice, and 2) to determine effects of 30% energy restriction
for 10 weeks on gene expression in adenoma tissue of Min mice. Adenoma number and
size in the small intestine and colon were measured to determine adenoma formation and
growth. Complimentary DNA based arrays were used to determine expression of 1,200
mouse genes in small intestinal tumor tissue of control and energy restricted mice. The
number and diameter of adenomas in the small intestine in the restricted group at times 3,
6 and 10 weeks were significantly lower than in the control group (P<0.05). In the colon,
the changes in adenoma number and volume induced by energy restriction were not
different compared to controls (P>0.05). Of 1,200 genes studied, 8 were overexpressed
and 13 were underexpressed in adenoma tissue of energy restricted mice compared to ad
libitum fed mice. Four of the underexpressed genes were oncogenes and 4 of the
overexpressed genes were immune function proteins. Conclusions: Energy restriction
reduces adenoma number and size in the small intestine in the Min mouse but does not
cause a significant change in adenoma number and size in the colon. The reduced

adenoma number and size were associated with changes in expression of 21 genes.

6]

INTRODUCTION

The effects of energy restriction on carcinogenesis have been recognized for some
time. In 1909, Moreschi was the first to report the effects of underfeeding on
carcinogenesis. He observed that sarcoma transplanted into mice grew more slowly as
less and less food was offered. In 1914, Rous and Tannenbaum showed that
underfeeding also inhibited the growth of spontaneous tumors. By 1943, Tannenbaum
had shown that energy restriction inhibited the growth of both spontaneous and induced
tumors. Forty years later three groups demonstrated that energy restriction reduced the
growth of azoxymethane-induced colon tumors in rats (Pollard et al., 1984; Klurfeld et
al., 1987; Reddy et al., 1987). This inhibition of cancer by calorie restriction appears to
be independent of the proportion of calories derived from dietary fat. In chemically
induced models of colon and mammary cancers, carcinogenesis was inhibited in 40%
calorie-restricted rats even though they were fed a diet containing a higher percentage of
calories from fat (Klurfeld et al., 1987).

Despite the abundance of evidence for an inhibitory effect of energy restriction on
cancer, the mechanisms underlying this effect are poorly understood. Several
mechanisms have been postulated to explain this effect, such as, increased DNA repair
activity, altered gene expression, depressed metabolic rate and reduced oxidative stress.
Other proposed mechanisms suggest that energy restriction elicits its effects by altering
the hormonal balance in the animal. Energy restriction has been shown to stimulate the
pituitary-adrenocorticotropic axis, which results in decreased levels of mitogenic and
reproductive hormones (Weindruch and Walford, 1988; Ames and Shigenaga, 1994;

Leakey et al., 1994). This energy restriction-induced switch from a reproductive pattern

62

of life to one emphasizing repair and maintenance functions has been suggested to reduce
tumor incidence by lowering the metabolic rate, hence reducing the production of
metabolic by-products that could cause nuclear damage. There is evidence that cell
proliferation is decreased, and cell death by apoptosis is increased, in energy restricted
animals (Albanes et al., 1990; James et al., 1998). Energy restriction reduced colon
weight, the number of cells engaged in DNA synthesis, and the total number of crypts.
The reduction in cell divion at any given time was suggested to impart resistance against
induction of colon cancer in these animals (Albanes et al., 1990). A chronic dietary
restriction was shown to increase the spontaneous apoptotic rate and to decrease cell
proliferation rate in hepatocytes of 12-month old B6C3F1 mice (James et al., 1998).

Cellular proliferation and death are controlled by the expression of genes that regulate
cell division cycle. Energy restriction has been shown to alter gene expression. Zhu et al.
(1999) have studied the effects of energy restriction on expression of genes that are involved in
cell cycle progression in chemically induced mammary carcinogenesis. Energy restriction, in a
dose dependent manner, up—regulated p27/kip], a gene product associated with cell cycle
growth arrest, and down-regulated cyclin D1, a protein that combines with cyclin-dependent
kinases to promote phosphorylation of retinoblastoma protein and progression of the cell
through the cell cycle. Lee et al. (1999) have reported changes in gene expression in mouse
muscle cells in response to aging and energy restriction. Their study showed that age-related
changes in gene expression were partially reversed by energy restriction. For example genes
involved in energy metabolism were down-regulated in aged animals but were up regulated in
energy restricted animals. Similarly, genes involved in protein metabolism and biosynthesis

were up-regulated as a result of energy restriction. In contrast, the genes involved in stress

63

response, e. g., the heat shock response genes, DNA-damage-inducible genes, and oxidative
genes were up—regulated in aged animals, but were down-regulated in energy restricted animals
(Lee et al, 1999). These studies provide evidence for the modulation of gene expression by
energy restriction. The effect of energy restriction on expression of a wide range of genes in
the Min mouse model has not been reported.

In the Min mouse, a model for human colon cancer, a germline mutation in a single
allele of the Apc gene leads to formation of multiple adenomas in the small intestine. Tumors
appear in the small intestine of Min mouse very early in life with intestinal adenomas present
as early as 3 weeks after birth. Tumor number increases with age and plateaus at about 12
weeks after birth (Kang et al, 1999). However, the focal nature of adenomas and the presence
of tumor-free regions in Min mice suggest that a single Apc mutation is not sufficient for tumor
formation and further mutations are required to form an adenoma. We reported earlier that 20%
energy restriction for a 11-week period significantly reduced the number of adenomas in the
small intestine of Min mice. Thus, energy restriction may reduce adenoma number by delaying
or preventing the occurrence of subsequent mutational events. The present study examined
adenoma formation and development in Min mice after 3, 6 and 10 weeks of 30% energy
restriction. In addition, complimentary DNA—based array technology was used to identify

genes whose expression was altered by energy restriction.

MATERIALS AND METHODS
Experimental design
Fifty-six female Min mice were assigned to the 30% energy restricted (ER) or ad libitum
fed control (CT) groups at 3-5 weeks of age. Eight mice from each group were killed at
3, 6 and 10 weeks after treatment was begun and tumor data were collected. Two of the
mice from each group killed after 10 weeks of treatment had total RNA collected for

cDNA rrricroarray analysis. Body weight were measured weekly during the 10 week trial.

Animal Diets

Feed intake in the CT group was measured twice weekly and feed intake for the restricted
group was adjusted accordingly to provide 70% of the calories consumed by CT group.
All mice were individually housed in wire bottom cages to facilitate measurement of feed
intake. The diet for the energy restricted group was formulated to be lacking only in
energy from carbohydrates (cornstarch). The energy restricted group and the ad libitum

fed group received an equal amount of all other nutrients (Table 1).

Tissue Preparation

Mice were killed by C02 inhalation, and small intestines and colons were removed. The
small intestines and colons were cut open longitudinally and cleaned with water. The
colons were pinned flat, the small intestines were flattened between sheets of filter paper,
and both were fixed overnight in 4% 4 buffered formalin (pH 7.4). The tissues were
stained in a 0.2% solution of methylene blue and stored in 1% formalin in phosphate

buffered saline until the tumors were counted and measured. Counting and measuring of

65

Table 1. Composition of diets fed to control and energy restricted groups and the mean
daily intake of each component.

 

 

 

 

 

 

 

 

 

 

 

 

Ad Libitum Diet Calorie-Restricted Diet

g/kg Mean daily g/kg Mean daily
Diet In redients Intake (g) Intake (g)
Cornstarch 399.4 1.28 279.6 0.7
Casein 222.2 0.71 222.2 0.6
Sucrose 100 0.32 100 0.25
Soybean oil 166.7 0.53 166.7 0.42
Solka floc 55.5 0.18 55.5 0.13
Mineral mix (AIN-93G-MX) 38.89 0.12 38.8 0.09
Vitamin mix (AlN-93-VX) l 1.11 0.04 11.11 0.03
L-Cysteine 3.33 0.01 3.33 0.01
Choline bitartarate 2.78 <0.01 2.78 <0.01
T-butylhydroquinone 0.02 <0.01 0.02 <0.01
Calories/g 4.25 4.28
Calories consumed/da 13.6 9.4

 

66

 

 

 

tumors were done by a person blinded to the treatments. To harvest RNA for rrricroarray
analysis, small intestines were opened longitudinally, emptied of their contents and rinsed
in water. The whole tissue was examined under a dissecting microscope to identify
tumors. Tumor tissue was separated from the underlying muscle and connective tissue
layer with the tip of a pasteur pipette and dispensed into microfuge tubes containing
Trizol LS reagent (Gibco BRL, Rockville, MD). Trizol LS reagent is a mono-phasic
solution containing phenol and isothiocyanate. RNA was isolated by the single step RNA
isolation method developed by Chomczynski and Sacchi (1987). In this experiment, the
tumors from animals in each treatment group were pooled to ensure sufficient amounts of

RNA.

RNA Isolation

Tumor tissues from control and energy restricted mice were lysed in Trizol LS reagent by
sonication to ensure complete dissociation of nucleoprotein complexes. Total RNA was
obtained by following the instructions for the use of Trizol reagent provided by Gibco
BRL (Rockville, MD). The clear RNA pellet was washed in ethanolzwater solution and

dissolved in RNase-free water.

cDNA Synthesis and Hybridization

Five micrograms of mRNA was used to synthesize cDNA using MMLV reverse
transcriptase (Gibco BRL, Rockville, MD) and oligo dT primer. The membranes are pre-
hybridized with ExpressHyb (Clontech, Palo Alto, CA) and salmon sperm DNA for 30

minutes at 68°C. Non-specific binding was reduced by the addition of 5 ug of human Cot

67

DNA to the hybridization mixture. The [33P]dATP labeled probes from tumor tissue
from energy restricted and control animals were hybridized to pre-wet membranes
overnight at 42 °C in ExpressHyb solution. The membranes were washed 4x30 minutes
with 2xSCC/ 1% SDS at 68 °C and 2 washes with 2xSCC/0.5%SDS also at 68 °C. To
detect the [33 P] label, the phosphor screen was exposed from 1 hour to overnight. The
label was visualized and quantitated using a phosphorimager and comparisons of gene
expression across groups were made.

Numerical data was normalized by dividing each value by the overall mean of
values derived from that membrane. Fold expression was obtained by taking ratios of the

values for the two treatments.

68

RESULTS
M
The growth of the ad libitum fed and energy restricted mice are shown in Figure l. The
weights of the restricted mice were consistently and significantly lower than those of the
ad libitum fed mice. The ad libitum fed control mice gained weight steadily during the
first eight weeks of treatment and then began to lose weight during the final two weeks of
the treatment. The restricted mice maintained their weight during the first 4 weeks and

gained weight slightly in the following six weeks.

Small Intestinal Adenoma number

The number of adenomas increased during the first six weeks for both treatment groups.
The increase, however, was more pronounced in the ad libitum fed control mice. The
number of adenomas in the 30% energy restricted group at 3, 6 and 10 weeks was
statistically less than the number of adenomas in the control group (P<0.05) (Figure 2).

The energy restricted group had 33% fewer tumors than the ad libitum fed mice.

Small Intestinal Adenoma Diameter

Changes in small intestinal adenoma diameter at 3, 6 and 10 weeks in the control and
restricted groups are shown in Figure 3. There was a steady increase in the diameter of
the adenomas in the control group from 0 to 10 weeks, whereas, the adenoma diameter in
energy restricted mice remained unchanged. Overall treatment effect was significant at

p<0.05. Effect of time, however, was not significant.

69

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Colon Tumor Number

The numbers of colon tumors at 3, 6 and 10 weeks of treatment were not significantly
different (P=O.7l) between the control and energy restricted groups (Figure 4). As there
was no overall treatment effect, comparisons at the different time points were not

conducted.

Colon Tumor Volume

Tumor volume in colons of the ad libitum-fed mice appeared to rise sharply after the third
week of treatment and remained high for the remainder of the experiment (Figure 5).
Colon tumor volume for the energy restricted group, however, remained steady
throughout the experiment. The differences between the control and the energy restricted

group were not significant (P<0.07).

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75

Gene Expression

A comparison of mRNA levels in tumor tissue from the small intestine of energy
restricted animals and control ad libitum-fed Min mice revealed that 30% energy
restriction is associated with changes in mRNA levels. Out of 1,200 genes studied, 11
genes (0.9%) in tumors from energy restricted showed a greater than 1.6—fold increase in
expression compared to tumor tissue from ad libitum fed control mice. Over-expressed
genes and their functional categories are listed in Table 2. The over-expressed genes
include genes associated with immune function, growth factors and transcription factor,
and genes related to apoptotic pathways.

Thirteen out of 1,200 genes (1%) showed a greater than 1.7-fold decrease in
expression in tumors from energy restricted mice compared to tumors from control mice.
The genes that were expressed at a lower level in the energy restricted mice and their
functional categories are listed in table 3. Among the genes that are underexpressed are 4

proto-oncogenes or oncogene related proteins and one DNA excision repair gene.

76

Table 2. Over-expressed genes in tumor tissue of energy restricted micel.

Fold T i Functional Category —
Ex . ression

i isabled homolog 2 (Drosophila) . Negative growth regulator

IlPEPTIDYL-PEPTIDASE l PRECURSOR (DPP-l) . Lysosomal protease
CATHEPSIN C) (CATHEPSIN J) (DIPEPTIDYL

: RANSFERASE)

gt ibroblast growth factor 15 (FGF15) . Regulator of cell division

; ytoplasmid dynein light chain 1; protein inhibitor of . Neuronal protective protein
- euronal nitric oxide synthase (mPIN)
nasic fibroblast growth factor receptor 1 precursor . Role in wound healing
BFGF-R; FGFR1); MFR; FLG
amma interferon-induced monokine precursor . Immune Function
, MIG); M119
’ D14 monocyte differentiation antigen precursor; . Immune Function
i. PS receptor (LPSR); myeloid cell-specific leucine-
rich flycoprotein
l F-kappa-B transcription factor p65 subunit (NF-kB . Immune Function
[:5 ; relA; NFKBS

 

 

 

 

 

 

 

 

 

 

1Expression of 1,200 genes was analyzed using cDNA arrays in tumor tissues obtained
from ad libitum-fed and energy restricted Min mice. Fold increase in expression was
obtained by taking the ratio of normalized levels of RNA corresponding to each gene in

cad-libitum fed (control) and energy restricted animals.

77

Table 3. Under-expressed genes in tumor tissue of energy restricted mice].

proto-oncogene;

(Heparin- factor precursor

Growth Factor 8) (Osteoblast
Factor 1) (OSF- (Heparin-Binding

gene
of protein (AES); enhancer of split

during development, mutated
in congenital disorder,

 

]A decrease in expression of these genes was found as a result of energy restriction of the
animals. Fold decrease in expression was obtained by taking the ratio of normalized

levels of RNA corresponding to each gene in energy restn'cted animals and ad libitum-fed

(control) animals.

78

DISCUSSION

The majority of adenomas in Min mouse occur in the small intestine, therefore,
the anti-neoplastic effects of energy restriction were more apparent in this part of the
intestine. In our experiment, the number of small intestine adenomas peaked at
approximately 9-12 weeks of age in the ad libitum-fed control mice. Kang et al (1999)
reported an increase in tumor number with age until about 12 weeks after birth.
Adenoma diameter, however, increased linearly until the termination of the experiment
(age 13-15 weeks). The slight drop in weight of the ad libitum-fed mice beginning at
week 8 of treatment was likely due to intestinal bleeding from the large number of
adenomas.

Weight gain in the energy restricted mice was nominal. Small intestine adenoma
number increased during the first two weeks of energy restriction (32 adenomas per
mouse) with no further increase with time of treatment (29 adenomas per mouse). The
difference in number of small intestine adenoma between the ad libitum-fed and energy
restricted groups increased with length of treatment. At 6 weeks of treatment there were
29% fewer adenomas in energy restricted mice and at 10 weeks of treatment there were
38% fewer adenomas in energy restricted mice. We have observed a similar degree of
inhibition (36% decrease) of adenoma formation in the Min mouse with a 20% energy
restriction in an earlier study (Mridvika et al., submitted).

Anti-carcinogenic effects of energy restriction have been well documented in
spontaneous tumors and azoxymethane-induced colon tumors (Reddy et al., 1987).
Several mechanisms have been postulated to explain the anti-cancer effects of energy

restriction. Some of the proposed mechanisms include increased capacity for DNA

79

 

repair, decreased metabolic rate, decrease in oxidative stress, and changes in gene
expression. Zhu et al. (1999) reported that energy restriction, in a dose dependent manner,
up-regulated p27/kipl, a protein associated with cell cycle growth arrest, and down-
regulated cyclin D1, a protein that promotes the progression of cells through the cell
cycle. Changes in gene expression by energy restriction have also been reported by Lee et
al. (1999). In the 6,000+ genes studied, 1.8% of the genes either increased or decreased
expression as a function of aging in mouse gastrocnernious mucsle (Lee etal., 1999).
Upon 24% energy restriction, a large number of these changes were completely reversed
and a significant number were partially reversed. The expression of a similar number of
genes (1.9%) was altered in intestinal tumors in our study.

The progression of normal-appearing intestinal epithelium to adenomas most
likely resulted from molecular changes that facilitate the progression of tumorigenesis.
These changes in the Min mouse are beginning to be elucidated including the loss of
wild-type allele of the Apc gene, the possible increased expression of additional
oncogenes, and/or decreased expression of tumor suppressor genes. Inhibition of
tumorigenesis in the small intestine by energy restriction was accompanied by decreased
expression of 4 oncogenic proteins. Jun B and c-jun proto-oncogene (transcription factor
AP-l; AH119) are immediate early genes that are induced during GO/Gl (Bravo, 1999).
These genes encode for molecules that can regulate the expression of other genes whose
products are essential for progression through the G1 phase of the cell cycle. L-myc is a
proto-oncogene protein from the myc family of oncogenes. Wnthb overexpression has
led to formation of mammary tumors in mice (Lane et al., 1997) and behaves like a proto-

oncogene leading to formation of tumors in transgenic mice.

80

Energy restriction slows metabolism, which reduces the production of potentially
toxic metabolic by-products of, which could cause macromolecular damage. The two-
fold decrease in the expression of XPG DNA repair protein suggests a reduction in the
need to repair damaged DNA. A similar argument has been offered by Lee et al. (1999),
for the down regulation of repair enzymes in energy restricted mice in their aging
experiments. PCNA, an auxillary protein for one of the DNA polymerase enzymes
(Bravo, 1987), is also down-regulated by two fold. This down-regulation of PCNA is
consistent with a slower metabolic rate and a smaller adenoma size observed in the
energy restricted group.

A gene that exhibited the greatest increase in expression as a result of energy
restriction was disabled homolog 2 (Dab 2), a mammalian structural homolog of
Drosophila disabled (Dab). Disabled homolog 2 is a mitogen-responsive protein that is
thought to be a negative regulator of growth as its expression is lost in ovarian cancer (Xu
et al., 1998). Dab 2 may also regulate Ras pathways by competing with other molecules
that bind Ras. Dipeptidyl-peptidase 1 (DPP I) precursor was also up-regulated in energy
restricted animals. DPP I, a lysosomal cysteine proteinase is important in the intracellular
degradation of proteins and is a central coordinator for activation of many serine
proteinases in immune/inflammatory cells (Rao etal., 1997).

It is noteworthy that 50% of the genes that were up-regulated as a consequence of
energy restriction are related to immune function. Basic fibroblast growth factor receptor
1 precursor (BFGF-R; FGFRI) is a potent mitogenic and chemotactic factor for
endothelial cells and fibroblasts (Akimoto et al., 1999) and may play an important role in

scar healing. The gamma interferon-induced monokine precursor (MIG) is a member of

81

the platelet factor 4-interleukin-8 cytokine family that is expressed in murine
macrophages specifically in response to IFN-gamma. Wong et al. (1994) also reported
that MIG is overexpressed in energy restricted animals. CD14 monocyte differentiation
antigen precursor; LPS receptor (LPSR) is responsible for the activation of B cells and
hence forms an integral part of immune function. NF-kappa-B transcription factor p65
subunit (NF-kB p65) is yet another immune molecule up-regulated by energy restriction.

An immune modulatory effect of energy restriction has been reported. Most
studies have suggested an impaired function resulting from weight loss induced by energy
restriction (Nieman et al., 1998; Shi et al., 1997). However, others have suggested an
immunostimulatory effect of energy restriction (Lim et al., 2000). The parameters of
immune function studied in these cases are probably responsible for the conflicting
results.

Energy restriction in Min mice for a period of 10 weeks changed expression of
genes in ways that slow tumor growth. An overall pattern emerging from this experiment
was a decreased expression of oncogenes and growth factors, an increase in the
expression of genes that slow down cell division, and a marked increase in genes
involved in immune function. These results are consistent with previous studies that

suggest that energy restriction reduces the need for DNA repair.

82

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