IMPACT OF ANTHOCYANINS AND DIETARY
MIN/+
CARBOHYDRATES ON INTESTINAL TUMORIGENESIS IN APC
MICE
By
Ryan Walker

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Human Nutrition – Master of Science
2013

ABSTRACT
IMPACT OF ANTHOCYANINS AND DIETARY CARBOHYDRATES ON INTESTINAL
MIN/+
TUMORIGENESIS IN APC
MICE
By
Ryan Walker
Consumption of anthocyanins reduces the development of
intestinal tumors in Apc

Min/+

mice, whereas diets rich in sucrose

increase intestinal tumor development. The aim of this study was
to determine the efficacy of anthocyanins to reduce intestinal
tumorigenesis in Apc

Min/+

mice consuming diets rich in sucrose and

other dietary carbohydrates. Mice were assigned to six different
modified AIN-93G diets, which contained different carbohydrate
sources (starch, sucrose, or glucose) and different doses of
anthocyanins (0 or 1500 mg/kg) in a factorial design. To enhance
tumor development, mice were injected for three consecutive weeks
with azoxymethane (8 mg/kg) prior to dietary intervention. Mice
consuming sucrose showed the largest colonic adenomas compared to
mice consuming anthocyanin-sucrose, anthocyanin-glucose, glucose,
and starch (P<0.05). These results demonstrate that anthocyanins
and different carbohydrates influence intestinal adenoma
development in Apc

Min/+

mice; however, additional research is

required to determine the mechanisms for the observed effects.

ACKNOWLEDGMENTS

I would like to thank Dr. Les Bourquin for his advice,
encouragement, support, and open-mindedness to my research
and career plans. I would also like to thank my committee
members, Drs. Muralee Nair and Jenifer Fenton, for
supporting this research. My lab partner, Loan Cao, played
an important role in this research with her advice and
assistance. Additionally, I’d like to thank Connel
Chinganda, Sharon Hooper and Eric Gurzell for their
assistance during my busy summer months; and most
importantly the undergraduate students: Andrew Morton, Greg
Jones, Jenn Cooke, Vernon Lalone, Brandon Goller, and
Allison Chan. These students played a crucial role in
completing this research. Finally, I would like to thank
Dr. Beth Rondini for her technical assistance and for being
a listening ear.

iii

TABLE OF CONTENTS

LIST OF TABLES...............................................v
LIST OF FIGURES............................................vii
INTRODUCTION.................................................1
REVIEW OF THE LITERATURE.....................................4
1. Incidence of Colorectal Cancer.......................4
2. Genetic and Molecular Basis of Colon Cancer..........5
3. Inflammatory Mechanism of Colon Cancer...............9
4. NSAIDs as a Colon Cancer Treatment..................13
5. Dietary Factors Associated with Colon Cancer........14
6. Dietary Factors and Positive Health Outcomes........20
7. Polyphenols.........................................21
8. Bioavailability of Anthocyanins.....................23
9. Anthocyanins as Anti-inflammatory Compounds.........26
10. Anthocyanins as Antioxidant Compounds..............27
11. In Vitro Studies Using Human Cancer Cell Lines.....28
12. Plant Phenolics in Animal Studies..................32
Min/+
13. Apc
Mouse Model and Azoxymethane..............34
RATIONALE AND SPECIFIC AIMS.................................36
MATERIALS AND METHODS.......................................40
1. Animals.............................................40
2. Diets...............................................42
3. Weight and Body Composition.........................44
4. Sample Collection...................................44
5. Quantification of Intestinal Adenomas...............45
6. Statistical Analyses................................46
RESULTS.....................................................48
1. Body Weights and Body Composition...................48
2. Small Intestinal Adenomas...........................49
3. Colonic and Cecal Adenomas..........................52
DISCUSSION..................................................70
SUMMARY AND CONCLUSIONS.....................................82
REFERENCES..................................................85

iv

LIST OF TABLES

Table 1.

Composition of Diets...........................44

Table 2.

Total small intestine adenoma number, average
Min/+
size, and burden in Apc
mice fed diets
without (-) or with (+) 1500 ppm anthocyanins:
(-) cornstarch, (-) sucrose, (-) glucose, (+)
cornstarch, (+) sucrose, or (+) glucose........63

Table 3.

Proximal small intestine adenoma number, average
Min/+
size, and burden in Apc
mice fed diets
without (-) or with (+) 1500 ppm anthocyanins:
(-) cornstarch, (-) sucrose, (-) glucose, (+)
cornstarch, (+) sucrose, or (+) glucose........64

Table 4.

Medial small intestine adenoma number, average
Min/+
size, and burden in Apc
mice fed diets
without (-) or with (+) 1500 ppm anthocyanins:
(-) cornstarch, (-) sucrose, (-) glucose, (+)
cornstarch, (+) sucrose, or (+) glucose........65

Table 5.

Distal small intestine adenoma number, average
Min/+
size, and burden in Apc
mice fed diets
without (-) or with (+) 1500 ppm anthocyanins:
(-) cornstarch, (-) sucrose, (-) glucose, (+)
cornstarch, (+) sucrose, or (+) glucose........66

Table 6.

Colonic adenoma number per mouse, adenoma number
per adenoma bearing mouse, and percent incidence
Min/+
in Apc
mice fed diets without (-) or with (+)
1500 ppm anthocyanins: (-) cornstarch, (-)
sucrose, (-) glucose, (+) cornstarch, (+) sucrose,
or (+) glucose..................................67

Table 7.

Average colon adenoma size and adenoma burden in
Min/+
all Apc
mice fed diets without (-) or with
(+) 1500 ppm anthocyanins: (-) cornstarch, (-)
sucrose, (-) glucose, (+) cornstarch, (+) sucrose,
or (+) glucose..................................68

v

Table 8.

Average colon adenoma size and adenoma burden in
Min/+
adenoma bearing Apc
mice fed diets without
(-) or with (+) 1500 ppm anthocyanins: (-)
cornstarch, (-) sucrose, (-) glucose, (+)
cornstarch, (+) sucrose, or (+) glucose.........69

vi

LIST OF FIGURES

Figure 1. Chemical Structures of Common Anthocyanidins....23
Figure 2. Chemical Structures of Anthocyanin Metabolite...31
Figure 3. Experimental Design.............................41
Figure 4. Chemical Structures of Anthocyanins from Tart
Cherries........................................43
Figure 5. Weekly average body weights of wild type C57BL/6J
Min/+
and Apc
mice..............................55
Figure 6. Final body weights of pooled male and female wild
Min/+
type C57BL/6J and Apc
mice..................56
Figure 7. Final body weights of male and females by wild
Min/+
type C57BL/6J and Apc
status...............57
Figure 8. Weekly average body weights of male and female
Min/+
wild type C57BL/6J and Apc
mice............58
Figure 9. Carbohydrate effect on pooled C57BL/6J and mice
Min/+
Apc
mice weekly mean body weights..........59
Min/+

Figure 10. Weekly average female Apc
mice body weights
when fed diets without (-) or with (+) 1500 ppm
anthocyanins: (-) cornstarch, (-) sucrose, (-)
glucose, (+) cornstarch, (+) sucrose, or (+)
glucose.........................................60
Min/+

Figure 11. Weekly average male Apc
mice body weights
when fed diets without (-) or with (+) 1500 ppm
anthocyanins: (-) cornstarch, (-) sucrose, (-)
glucose, (+) cornstarch, (+) sucrose, or (+)
glucose.........................................61
Figure 12. Mean body fat percentage of wild type C57BL/6J
Min/+
and Apc
mice...............................62

vii

Figure 13. Mean lean body mass percentage of wild type
Min/+
C57BL/6J and Apc
mice......................62

viii

INTRODUCTION

Approximately one out of two American men and women
will develop cancer during their lifetime. The risk of
developing colorectal cancer, the second leading cause of
cancer related deaths, is approximately five percent
(National Cancer Institute, 2013). Nearly one-third of all
cancer related deaths have been attributed to inappropriate
diet and lack of physical activity (American Cancer
Society, 2013). However, colorectal cancer mortality rates
have declined over the previous two decades, and this
decline has been linked to improved cancer screening and
earlier detection methods.
The American Cancer Society recommends limited
consumption of red meat, processed meats, calorie-dense
foods, refined carbohydrates, and sugar-sweetened
beverages; and emphasizes increased consumption of fruits,
vegetables, and whole grains. Epidemiological data support
these recommendations. For example, people who consumed
diets higher in red meat, processed meat, refined grains,
and sugar-sweetened beverages had a higher risk of
developing certain cancers (American Cancer Society, 2012).
In contrast, people who regularly consumed more fruits and

1

vegetables had a lower risk of developing cancer (Miller et
al., 2010).
The dietary components of calorie–dense diets most
commonly associated with increased colorectal cancer risk
are sucrose and refined carbohydrates. Both have been
positively correlated to increased risk in several
epidemiological studies (Franceschi et al., 2001; Bostick
et al., 1994; Slattery et al., 1997).
Animal experiments also have demonstrated a
relationship between sucrose consumption and colorectal
tumorigenesis (Stamp et al., 1993; Caderni et al., 1993;
Caderni et al., 1997; Poulsen et al, 2001; Kristiansen et
al., 1995; Kristiansen et al., 1996; Wang, 2005; Wang et
al., 2009).
On the other hand, fruits and vegetables contain a
wide variety of phenolic compounds, which possess the
potential to act as chemopreventative agents due to their
strong antioxidant and anti-inflammatory activities (Blando
et al., 2004). For example, tart cherries are rich in
phenolic compounds called anthocyanins. Other fruits rich
in anthocyanins are blueberries, strawberries,
elderberries, and cranberries.
Evidence from cell culture, animal and human studies
have demonstrated anthocyanins are absorbed and act as

2

antioxidants and anti-inflammatory agents in vitro and in
vivo. However, the potential for anthocyanins to inhibit
cancer in animals fed diets rich in refined carbohydrates
has not yet been determined.
This study was designed to determine the influence of
anthocyanins on intestinal adenoma development associated
with refined sugar consumption in Apc
human colorectal cancer.

3

Min/+

mice, a model for

REVIEW OF THE LITERATURE

1. Incidence of Colorectal Cancer
Colorectal cancer is the third most commonly diagnosed
cancer, and the second highest cause of cancer mortality in
both men and women in the United States (American Cancer
Society, 2013). In 2012, the National Cancer Institute
estimated 143,460 new incidences per year in the U.S., of
which approximately 51,690 will be fatal. The lifetime risk
for developing colorectal cancer for American males and
females is 4.96 percent (National Cancer Institute, 2013).
Risk also increases with age, as 90 percent of all
colorectal cancer cases occur in people 50 years of age and
older. In addition to age, other risk factors include:
family history, genetic conditions, lack of physical
activity, alcohol consumption, and tobacco use (Centers for
Disease Control and Prevention, 2013). Specific dietary
components also have been negatively and positively
correlated to risk. Diets high in protein and fat (Potter
et al., 1993), and refined carbohydrates (Franceschi et
al., 2001; Bostick et al., 1994; Slattery et al., 1997)
have been linked to increased colon cancer risk, whereas
diets that regularly incorporate vegetables and fruit

4

reduce cancer risk (Terry et al., 1998; Terry et al., 2001;
Miller et al., 2010).

2. Genetic and Molecular Basis of Colon Cancer
Inherited genetic defects have been useful in
elucidating molecular mechanisms of carcinogenesis, and at
least 15 percent of colon cancers can be linked to
inherited genetic diseases. The two primary genetic
diseases associated with colorectal cancer are familial
adenomatous polyposis (FAP) and hereditary nonpolyposis
colorectal cancer (HNPCC). Familial adenomatous polyposis
is a dominantly inherited disease affecting approximately 1
in 10,000 people (Jasperson et al., 2010) and accounts for
one to three percent of colon cancer incidences (Burt,
2000). Patients with FAP develop hundreds to thousands of
adenomas during their 20’s and 30’s. Although the adenomas
are usually benign, the large number of adenomas leads to a
higher risk that some will progress into carcinomas
(Kinzler and Vogelstein, 1996).
Familial adenomatous polyposis is the result of a
mutation in the adenomatous polyposis coli (Apc) gene. The
Apc gene codes for a protein that is important for the Wnt
signaling pathway, intercellular adhesion and apoptosis
(Gryfe et al., 1997; Fearnhead et al., 2001). Adenomas can

5

develop when the normal Apc allele is lost or mutated.
Patients with FAP only have one normal Apc allele, and a
mutation can result in Apc gene inactivation (Burt, 2000).
Additionally, mutations or deletions in both copies of Apc
are commonly present in approximately 80 percent of
sporadic colorectal tumors (Markowitz and Bertagnolli,
2009; Fearnhead et al., 2001).
Carcinogenesis is a multistep process that involves
genetic or epigenetic alterations of multiple genes, and
colorectal cancers commonly have three or more genetic
alterations (Barrett, 1993; Calabrese et al., 2005). The
process of carcinogenesis involves initiation, promotion,
and progression (Pitot et al., 1981). Initiation is the
irreversible alteration of a cell and is commonly
associated with genetic mutations. Promotion is the clonal
expansion of mutated cells into visible benign tumors or
preneoplastic cells. Progression is the transition phase
when benign tumors or preneoplastic cells become malignant
cancers (Barrett, 1993). However, carcinogenesis is not a
linear mechanism involving a straightforward series of
genetic alterations. Rather, it is a result of multiple
alterations that affect tumor suppressor genes, protooncogenes, and DNA repair genes (Gryfe et al., 1997). Genes
commonly mutated in genetic or sporadic colon cancers are

6

Apc, Kristen rat sarcoma (Kras), deleted in colorectal
carcinoma (Dcc), and the tumor protein p53 (TP53)
(Vogelstein et al., 1988).
The Apc gene is a tumor suppressor gene located on the
5q12 chromosome and codes for a protein that is important
for the Wnt signaling pathway, intercellular adhesion and
apoptosis (Gryfe et al., 1997; Fearnhead et al., 2001).
Mutations in the Apc gene can result in the loss of the APC
protein in the Wnt signaling pathway, resulting in the
accumulation of β-catenin in the cytoplasm. Liberated βcatenin translocates to the nucleus and activates genes
involved in cellular growth and proliferation such as
cyclin D1 and c-myc (Kwong and Dove, 2009).
The Kras gene is a proto-oncogene, which encodes a
GTPase protein involved in proliferation, apoptosis, cellcell adhesion, and cell motility. Cells with a mutated Kras
gene have characteristics such as poor cellular adhesion,
increased motility, increased growth, and invasiveness,
which are components of tumorigenesis (Pollock et al.,
2005).
The Dcc gene is located on chromosome 18q and
approximately 60 to 80 percent of all colorectal cancers
have been shown to lose this gene. It encodes a membrane
bound protein, which functions as a tumor suppressor by

7

signaling cell death via a caspase-dependent cell death
pathway (Mehlen and Fearon, 2004). Absence of Dcc fosters
an environment for enhanced cell survival.
Loss of the TP53 gene on chromosome 17p occurs in
approximately 70 percent of colorectal cancers. This gene
encodes a nuclear phosoprotein (p53) involved in activating
DNA transcription and DNA repair. Proteins produced by p53
activation are involved in cell cycle arrest and include
the p21 protein. The ability to arrest the cell cycle and
repair DNA damage is lost with mutations to the TP53 gene,
and can lead to cells with enhanced proliferation and the
ability to escape programed cell death (Gryfe et al.,
1997).
Enhanced proliferation, growth motility, invasiveness,
survival and immortality are biological capabilities
acquired throughout the multistep development of colorectal
cancer and are considered to be “hallmarks of cancer”
(Hanahan and Weinberg, 2011). The foundation of these
hallmarks is genetic instability, which fosters an
environment for increased accumulation of genetic
mutations. An additional hallmark, inflammation, has been
proposed by Colotta et al. (2009) as a key modulator of
genetic instability.

8

3. Inflammatory Mechanism of Colon Cancer
For more than a two hundred years scientists have
suspected a link between cancer and inflammation. In the
mid-1800’s, Rudolf Virchow hypothesized cancers originated
at sites of chronic inflammation. Virchow believed that
irritants, tissue injury, and inflammation enhanced cell
proliferation (Coussens and Werb, 2002). In itself,
enhanced cell proliferation does not cause cancer; however,
cells subjected to chronic inflammatory microenvironments
have been shown to acquire genetic mutations that can lead
to unregulated proliferation, survival, angiogenesis,
metastasis, and reduced response to therapeutic agents
(Colotta et al., 2009).
Inflammation is linked to the multistep carcinogenesis
process by acting either as an initiator or promoter of
tumors. As an initiator, an inflammatory microenvironment
can increase the rate of genetic mutations. Inflammatory
cells can also induce DNA damage by producing reactive
oxygen species (ROS) or cytokines, such as tumor necrosis
factor-α (TNF-α), which stimulate ROS production
(Grivennikov et al., 2010).
As a promoter, inflammation activates transcription
factors such as nuclear factor kappa-light-chain-enhancer
of activated B cells (NF-κB), which stimulates initiated

9

cells to turn on genes that stimulate cell proliferation,
invasiveness, angiogenesis, and survival (Grivennikov et
al, 2010; Sandur et al., 2009).
A selection of key inflammation regulators in colon
cancer include proinflammatory cytokines such as TNF-α and
interleukin-6 (IL-6), and the nuclear transcription factor
NF-κB. Cyclooxygenase-2 (COX-2), an enzyme responsible for
prostaglandin production, is also a key component in
inflammatory responses and a target for colorectal cancer
treatment.
NF-κB is activated by infectious agents, cytokines, and
danger signals secreted by necrotic cells (Karin, 2006).
Cell-proliferating genes regulated by NFκB include: TNF-α,
IL-6, interleukin-1 (IL-1), and cyclin D1 (Sethi et al.,
2008). Activation of NF-κB also promotes cell survival
through the activation of genes that negatively regulate
apoptosis. A selection of these genes includes B cell
lymphoma-2 (Bcl-2) and B cell lymphoma extra large (BclxL). These proteins suppress the release of cytochrome C
from the mitochondria. Cell invasiveness is mediated
through NF-κB by the activation of matrix metalloproteinases
(MMP), which are also proangiogenic (Sethi et al., 2008).
In general, the tumor promoting effect of NF-κB has
classically been described to be activated by viruses,
10

bacterial infections, and proinflammatory cytokines. These
agents activate the IκB kinase (IKK) complex, which is
composed of three subunits: IκB kinase-α (IKK-α), IκB
kinase-β (IKK-β), and IκB kinase-γ (IKK-γ). The activated
IKK complex phosphorylates NF-κB inhibitory subunits called
IκBs, which liberates NF-κB to translocate to the nucleus
for gene transcription (Karen and Greten, 2005).
Tumor necrosis factor-α is a cytokine and a regulator
of host defense and inflammation. It is produced by
monocytes, macrophages and T-cells to stimulate
proliferation, differentiation and inflammation in many
different types of cells. Tumor necrosis factor-α is also
produced by tumor cells and can act as an autocrine or
paracrine agent (Aggarwal et al., 2006). When TNF-α binds
to its receptors, TNFR1 or TNFR2, inflammation and cell
survival pathways are activated via NF-κB and caspase 8 in
colonic epithelial cells (McConnell and Yang, 2009). NF-κB
also activates genes for IL-6, inducible nitric oxide
synthase, and aldose reductase (AR), which are involved in
inflammatory mechanisms. Over expression of COX-2, mediated
by TNF-α, is dependent on NF-κB (Tammali et al., 2007). In
tumorigenesis, TNF-α is linked to unregulated

11

proliferation, survival, invasion, and angiogenesis in
several types of epithelial cells (Aggarwal et al., 2006).
The COX-2 enzyme is a cytoplasmic protein that
catalyzes the synthesis of prostaglandins from arachidonic
acid (AA). In the initial enzymatic reaction, AA is
converted to prostaglandin G2 by COX-2 or Cyclooxigenase-1
(COX-1), which is subsequently converted to prostaglandin
H2, also by COX-2. Prostaglandin H2 is then converted to
several structurally related prostaglandins by
prostaglandin synthases. These include the eicosanoids:
prostaglandin E2 (PGE2), prostaglandin D2, prostaglandin
F2α, prostaglandin I2, and thromboxane A2. These
eicosanoids then act through specific G-protein-coupledreceptors to maintain normal physiological processes, which
include immune function, maintenance of gastrointestinal
mucosa, blood clotting, and renal homeostasis. However,
prostaglandins also promote inflammation, swelling, pain,
and fever (Greenhough et al., 2009). Baseline levels of
prostaglandins are maintained by COX-1 and have been shown
not to be up regulated in colonic tumors, whereas the COX-2
enzyme is regulated in colorectal cancer (Sano et al.,
1995).
Transcription of the COX-2 gene is activated by proinflammatory cytokines, which include IL-1 and TNF-α. Its

12

expression is increased at inflammatory sites, and over
expressed in colorectal cancer in 80 percent of carcinomas
and 40 percent of adenomas. COX-2 is believed to contribute
to colorectal cancer promotion by inducing resistance to
apoptosis, and stimulating angiogenesis and invasiveness
(McConnell and Yang, 2009).
Cyclooxygenase enzymes were first implicated in
colorectal cancer when patients with Gardner’s syndrome
were treated with Non-steroidal Anti-inflammatory Drugs
(NSAIDs) and displayed reduced numbers of adenomas after
treatment. This was also the first clinical study that
suggested NSAIDs could be used to treat colorectal cancer
(Waddell et al., 1989; Greenhough et al., 2009).

4. NSAIDs as a Colon Cancer Treatment
Data from epidemiological studies and laboratory
experiments demonstrate NSAIDS may be protective against
colorectal cancer. From 1982 to 1988, more than 662,000
adults participated in a prospective cohort study
investigating the relationship between aspirin use and
mortality. Colon cancer death rates decreased in both men
and women who frequently consumed aspirin. The relative
risk of death resulting from colon cancer with aspirin
consumption at least 16 times/month was 0.60 (95% CI: 0.40-

13

0.89) and 0.58 (95%CI: 0.37-0.90) for men and women,
respectively. The investigators concluded that regular
aspirin use might reduce colon cancer via the inhibition of
prostaglandins (Thun et al., 1991). In a second prospective
mortality study of more than 635,000 people by the same
research group, duration of aspirin use was also shown to
reduce the relative risk of developing colon cancer. Men
and women, adjusted for age, race, and sex, who consumed
aspirin at least 16 times/month for 1-9 years had a
relative risk of 0.71, whereas those who consumed it for 10
years or more with the same frequency had a significantly
lower relative risk of 0.36 compared to all nonusers of
aspirin (Thun et al., 1993).
In animal experiments, aspirin has been shown to
reduce colonic tumor incidence, burden and expression of
PGE2 in rats (Reddy et al., 1993). For example, the NSAIDs,
sulindac and piroxicam, protect against colon tumor
development in mice and rats (Moorghen et al., 1988;
Skinner et al., 1991; Pollard and Luckert, 1989; Reddy et
al., 1987).

5.Dietary Factors Associated with Colon Cancer
The western diet, a diet with high intakes of red
meats, sweets, processed foods and refined grains, has long

14

been suggested to influence development of colon cancer
(Fung et al., 2003). Epidemiological studies have
associated an increased risk for developing colorectal
cancer in people who consume western style diets. Using
dietary information collected over 20 years from more than
76,000 women, the western diet was shown to have a relative
risk for colon cancer of 1.46 (95% CI, 0.97-2.19) when the
highest and lowest quintiles of the western style diet were
compared. However, diets with higher intakes of fruits,
vegetables, legumes, fish, poultry, and whole grains were
only observed to have the possibility of being inversely
associated with colon cancer risk (Fung et al., 2003).
In a case control study conducted over three years in
California, Utah, and Minnesota, researchers demonstrated
an increased risk of colon cancer in men and women
consuming a western diet in 1,993 cases matched with 2,410
controls. In colon cancer cases diagnosed before age 67,
males had an odds ratio of 1.96 (95% CI: 1.22-3.15) and
women had an odds ratio of 2.02 (95% CI, 1.213.36)(Slattery et al., 1998).
However, in a Swedish study involving more than 61,000
women over approximately 10 years, a western style diet was
not identified as a risk factor for developing colon cancer
(Terry et al., 2001).

15

Fung et al. (2003) defined western style diets as
being higher in sweets and refined grains. Epidemiological
data support the association of carbohydrate consumption
and risk of colon cancer development. Franceschi et al.
(2001) demonstrated colon cancer risk was associated with
diets rich in high glycemic index foods with an odds ratio
of 1.7 (95% CI: 1.4-2.0) when the highest and lowest
quintiles were compared and data were adjusted for age,
sex, and other confounders.
Results from case control studies have also suggested
sugar consumption may be a risk factor for developing colon
cancer. Bristol et al. (1985) demonstrated the relative
risk of developing colon cancer increased by a factor of
3.6 (95% CI: 1.2-10.9) in persons consuming diets with
sugar consumption greater than 99 g/day. Additionally,
results of the Iowa Women’s Health Study cohort also
indicated that women who consumed diets rich in sucrose had
an increased risk of colon cancer (Bostick et al., 1994).
In a study conducted in India, heavy sugar consumption
also was shown to be a colon cancer risk factor. Based on
dietary data collected using a food frequency
questionnaire, consumption of sugar was determined by a
subject’s interest or non-interest in sweet foods. A
positive colon cancer association was observed for those

16

who consumed sweets daily when compared to those who did
not like sweets (Nayak et al., 2009).
Finally, in a case control study investigating the
association between simple carbohydrates and risk of colon
cancer, high sucrose intake was associated with increased
colorectal cancer risk in men less than 67 years old. The
odds ratio when the highest and lowest quintiles were
compared was 1.59 (95% CI: 1.07-2.37). Men and women
consuming diets with a high glycemic index were also
observed to have a greater risk for proximal colon tumors
with odds ratios of 1.58 (95% CI: 1.06-2.36) for men and
1.72 (95% CI: 1.11-2.67) for women when the highest and
lowest quintiles for glycemic index from dietary sugars
were compared (Slattery et al., 1997).
However, other case-control studies have shown a less
significant relationship between sugar consumption and
colon cancer (La Vecchia et al., 1988; Michaud et al.,
2005).
Experiments using animals have been useful in
confirming the results elucidated from epidemiological
studies. For example, results from animal studies have
shown an association between sucrose intake and colon
cancer development. Researchers investigated the effect of
10 g/kg of body weight of sucrose, glucose, or fructose

17

consumption on colonic epithelial cell proliferation and
aberrant crypt foci number in CF1 mice injected with
azoxymethane (AOM). Increased aberrant crypt foci formation
was observed in mice given gavages of sucrose (18.4 ± 1.5)
and fructose (13.1 ± 1.8) compared to water (8.6 ± 1.1)
(Stamp et al., 1993).
Microadenoma (MA) development was also shown to
increase in AOM injected mice consuming cooked sucrose
compared to the control-uncooked diet (48 ± 16 vs. 28 ± 13
MA) (Corpet et al., 1990). Additionally, colonic crypt
proliferation and aberrant crypt foci were significantly
greater in rats consuming sucrose versus starch (Caderni et
al., 1993). In studies by Caderni, one showed that rats
consuming sucrose-rich diets had greater mucosal cell
proliferation than rats that consumed starch-rich diets
after 105 days. Treatment with the carcinogen 1,2dimethylhydrazine (DMH), which was used to increase colonic
cell proliferation, did not influence outcome in diets
containing either 46 percent sucrose or starch (Caderni et
al., 1991). In a subsequent study wherein rats were fed
diets containing 46 percent sucrose or starch for three
months, the number of colonic ACF were the same between the
groups, but ACF were larger in size in the sucrose group
(Caderni et al., 1997). Additionally, a mixed diet

18

consisting of sucrose and starch has also been shown to
enhance ACF development in rats (Poulsen et al, 2001;
Kristiansen et al., 1995; Kristiansen et al., 1996).
Although these diets contained mixed carbohydrates, the
study demonstrated that diets based on refined
carbohydrates stimulate cell proliferation.
In a study investigating colon tumor incidence and
burden in Apc

Min/+

mice, mice fed sucrose for 10 weeks did

not show significantly different colonic adenoma
development from mice fed starch. However, mice consuming
sucrose were observed to have greater cell proliferation in
the colon as determined by Ki-67 antigen labeling (Wang,
2005). However, in a 16-week follow-up study, sucrose, when
compared to starch, influenced proximal small intestinal
adenoma number and size, and colonic adenoma incidence
(Wang et al., 2009; Wang 2005). In another study using
Apc

Min/+

mice, colon tumor number, average size, burden and

incidence were not influenced by carbohydrates when
compared to mice fed diets containing primary carbohydrates
of starch, fructose, glucose, sucrose, or an equal glucose
and fructose mixture (Powell, 2011). It is important to
note that studies conducted by Powell (2011) and Wang
(2005) did not utilize a carcinogen, such as DMH or AOM, to
induce cell proliferation. Additionally, as noted by Powell
19

(2011), the Apc

Min/+

mouse is prone to developing a

significant number of small intestinal tumors, which lead
to morbidity and can limit the effectiveness of the model
developing colonic tumors with short diet protocols.

6. Dietary Factors and Positive Health Outcomes
While diets rich in carbohydrates have been associated
with increased risk of colorectal cancer, diets rich in
fruits and vegetables reduce this risk. For example, the
diets of more than 492,00 men and women were analyzed to
study associations between dietary patterns and colorectal
cancer in the National Institutes of Health American
Association of Retired Persons (AARP) Diet and Health
Study. Fruits and vegetables were associated with a
significantly lower risk of colorectal cancer in an ageadjusted multivariate-adjusted model for men with high
scores for fruit and vegetable consumption, but there was
no association observed in women (Flood et al., 2008).
However, association between fruit and vegetable
consumption was less consistent, and hence another group of
researchers conducted a pooled analysis of 14 cohort
studies. A total of 5,838 cases out of 756,217 men and
women were diagnosed with colon cancer during a period of
6-20 years of follow-up. The results showed a multivariable

20

relative risk of colon cancer of 0.91 (95% CI: 0.82-1.01)
when the highest and lowest quintiles of fruit and
vegetable intake were compared (Koushik et al., 2007).
In a meta-analysis, 28 case-control studies and 12
cohort studies were analyzed to determine whether fruits
and vegetables were protective against colon cancer. Of the
studies identified, 13 were excluded for insufficient
vegetable consumption data, and 21 were excluded for
insufficient fruit consumption data. The authors concluded
a moderate decreased risk of colorectal cancer in persons
consuming diets containing greater than 100 g/day of fruits
or vegetables. However, in their analysis, data derived
from the case-control studies demonstrated a significant
protective effect due to consumption of fruits and
vegetables, but a non-significant protective effect for the
cohort studies (Riboli and Norat, 2003).

7. Polyphenols
Anthocyanins are non-nutritive phytochemicals
belonging to the class of compounds called phenolics and
occur naturally in plants, flowers, fruits and vegetables
(Prior, 2003). Water-soluble and known for their bright
red, purple and blue colors, it has been hypothesized that
the purpose of anthocyanins in plants is to attract

21

pollinating insects and birds. Additionally, they are
indicators of ripeness in fruits, serve as plant defense
compounds, and reduce photo-oxidative stress by savaging
free radicals (Beckwith et al., 2004; Quina et al., 2009).
Anthocyanins are the most abundant dietary flavonoid
(Jing et al., 2008) of the 8000 or more phenolics that have
been identified (Pietta, 2000). Derived from the amino
acids phenylalanine and tyrosine, they consist of two
benzene rings joined by a three-carbon chain (Pietta,
2000). The basic structure is a flavillium cation with
rings labeled A, B and C (Figure 1). Anthocyanins occur
naturally as glycosides. In reference to anthocyanins, the
aglycones are classified as anthocyanidins, whereas the
glycosides are classified as anthocyanins. The most common
aglycone structures are cyanidin, delphinidin,
pelargonidin, malvidin, and petunidin (Figure 1) (Harborne,
1958; Wang and Stoner, 2008). Hundreds of anthocyanins have
been identified, with sugars conjugated to the C3 hydroxyl
group on the C ring. The most commonly conjugated sugars
are: glucose, galactose, rhamnose, xylose and arabinose
(Harborne, 1958; Wang and Stoner, 2008).

22

Figure 1. Chemical Structures of Common Anthocyanidins

Anthocyanidin

R1

R2

Cyanidin

OH

H

Delphinidin

OH

OH

H

H

Petunidin

OCH3

OH

Malvidin

OCH3

OCH3

Pelargonidin

8. Bioavailability of Anthocyanins
Bioavailability is defined as the fraction of an
orally administered dose that is digested, absorbed and
metabolized through normal metabolic pathways (McGhie and
Walton, 2007).

23

Animal studies have shown anthocyanins are absorbed as
intact glycosides. Their absorption can be detected as
early as 15 minutes after ingestion in the plasma of rats
(Miyazawa et al., 1999; Ichiyanagi et al., 2006). In rats
given doses of cyanidin-3-glucoside at 160 mg/kg or 320
mg/kg of body weight by stomach tube, plasma cyanidin-3glucoside was detected by high-performance liquid
chromatography at concentrations of 406 µg/L and 669 µg/L,
respectively. Cyanidin-3,5-diglucoside was also detected in
plasma, and plasma concentration was determined to be dose
dependent for both glycosides tested (Miyazawa et al.,
1999). The bioavailability in rats orally administered or
intravenously injected with individual bilberry
anthocyanins was determined to range between 0.61 to 1.82
percent, and 0.93 percent for an anthocyanin mixture
(Ichiyanagi et al., 2006).
In human studies, Murkovic et al. (2001) reported very
low anthocyanin absorption based on low urinary excretion
of the intact glycoside. However, Ohnishi et al. (2006)
demonstrated that six of the 12 cranberry anthocyanins they
identified were absorbed and excreted in urine, and
absorption was estimated to be 5 percent of the amount
consumed. In a study with tart cherries, twelve healthy
human volunteers ingested a relative dose of 45 or 90 whole

24

cherries in powder form to determine anthocyanin
bioavailability. Unmodified anthocyanins, methylated
anthocyanins and glucuronic acid-conjugated derivatives
were detected in plasma and urine samples. The unmodified
anthocyanins detected in plasma were cyanidin-3glucosylrutinoside and cyanidin-3-rutinoside (Kirakosyan et
al., 2010). Additionally, cyanidin-3-glucoside and
cyanidin-3,5-diglucoside were detected in the plasma of 12
human subjects who orally ingested anthocyanins from
elderberries and black currents. However, the agylcone,
cyanidin, was not detected (Miyazawa et al., 1999).
The absorption mechanisms for anthocyanins and
anthocyanidins have not been clearly delineated to this
point. However, unmodified blueberry and grape anthocyanins
are rapidly absorbed in rat stomachs (Talavera et al.,
2003; Passamonti et al., 2003). The acidic environment of
the stomach and anthocyanins’ interaction with
bilitranslocase has been hypothesized as a mechanism for
anthocyanin transportation and detection of intact
anthocyanin in rat plasma (Passamonti et al. 2002). In
studies investigating specific regions of absorption in the
gastrointestinal tract, the jejunum, but not the duodenum,
ileum or colon, has also been shown to be the site of
absorption of boysenberry derived cyanidin-3-glucoside in

25

Swiss mice (Matuschek et al., 2006). The authors concluded
the specific location of anthocyanin transport may indicate
a site of active transport. Additionally, the glucose
transporter (GLUT2), which is found on both the apical and
basolateral membrane of the small intestine, has been
demonstrated to transport anthocyanins. In Caco-2 cells,
treatment with anthocyanins was also shown to increase
GLUT2 expression (Faria et al., 2009).

9. Anthocyanins as Anti-inflammatory Compounds
Anthocyanins have been shown to be inhibitors of
cyclooxygenase enzymes, which are involved in the synthesis
of prostaglandins. Isomers of the enzyme, COX-1 and COX-2,
have different physiological functions. Cyclooxygenase-1 is
considered to act as a constitutive enzyme in many tissues
and is important for producing prostaglandins required for
maintaining the gastric mucosa, renal blood flow and
platelet function (Crofford, 1997; Greenhough et al.,
2009). Conversely, COX-2 is an inducible enzyme present in
most cells and is responsible for inflammatory responses
(Hawkey, 1999).
Several studies investigating the anti-inflammatory
properties of tart cherries have demonstrated that
anthocyanins inhibit COX enzyme activity. Balaton and

26

Montmorency tart cherry extracts were shown to inhibit COX1 activity by 84 and 91 percent, respectively, and COX-2
activity by 76 and 87 percent when tested at 250 µg/ml in a
COX enzyme activity assay (Mulabagal et al., 2009).
Anthocyanin mixtures from cherries and berries were also
shown to inhibit COX enzyme activity at 125 µg/ml, but to
varying degrees. Additionally, purified cyanidin was shown
to inhibit COX-1 and COX-2 activity by 38.7 and 46.8
percent, respectively, when tested at a concentration of 5
µmol/L. However, purified cyanidin glycosides, when tested
at 10 µmol/L, had limited COX inhibitory activity (Seeram et
al., 2001). Purified cyanidin also has been shown to
inhibit COX-1 and COX-2 activity at 90 and 60 mmol/L,
respectively, but was also effective at 15 mmol/L (Wang et
al., 1999). In all experiments, COX enzyme inhibition by
cyanidin was comparable to the non-steroidal antiinflammatory drugs (NSAIDs) used as controls.

10. Anthocyanins as Antioxidant Compounds
The antioxidant activity of anthocyanins has been
shown to be comparable to commercially available
antioxidants such as butylated hydroxytoluene and tertbutylhydroquinone (Wang et al., 1999). Additionally,
antioxidant capacity of anthocyanins was inversely related
27

to the number of sugar residues attached at the C3 position.
Therefore, the greater the number of sugars residues
attached resulted in less antioxidant capacity being
observed. Additionally, cyanidin, when compared to tart
cherry glycosides, demonstrated the greatest antioxidant
activity. This was attributed to the aglycone having a more
stable aryloxyl radical (Wang et al., 1999).
The antioxidant activity of anthocyanins and
anthocyanidins has been attributed to radical-scavenging
capacity of the catechol structure of the flavillium
cation’s B ring (Figure 1) (Pietta, 2000).

11. In Vitro Studies Using Human Cancer Cell Lines
Treatment of HCT 116 and HT 29 human colon cancer
cells with anthocyanins or cyanidin causes a dose-dependent
reduction in cell growth without cytotoxicity. The IC50
values in these experiments for a mixture of anthocyanins
were 260 and 585 µmol/L for HCT 116 and HT 29 cells.
However, the IC50 values for cyanidin demonstrated it was
more effective in inhibiting cell growth. The cyanidin IC50
values for HCT 116 and HT 29 cells were 85 and 63 µmol/L,
respectively (Kang et al., 2003). Similarly, Yi et al.
(2005) demonstrated cancer cell proliferation was inhibited
28

by blueberry extracts in HT 29 and Caco-2 cells. The
anthocyanin fraction resulted in the greatest growth
inhibition in HT-29 cells, with an IC50 of approximately 2550 µg/ml depending on the blueberry variety. With Caco-2
cells, blueberry anthocyanins inhibited cell growth with
concentrations as low as 15 µg/ml. Although anthocyanins and
anthocyanidins have been shown to inhibit cell
proliferation, their stability at physiological pH has been
debated. At a pH < 3, the primary tart cherry anthocyanidin
is the red flavylium cation (Seeram et al., 2001). As pH
increases, the blue colored quinonoidal structure forms due
to deprotonization of oxygen. Hydration of the flavylium
cation at higher pH leads to the formation of chalcones
with cis and trans structures. At physiological pH, the
primary bioactive compounds have been hypothesized to be
quinonoidal bases, hemiketals and chalcones (McGhie and
Walton, 2007).
The bioactive components of Balaton cherries are
cyanidin 3-glucosylrutinoside, cyanidin 3-rutinoside, and
cyanidin 3-glucoside. Since anthocyanins are unstable at
higher pH and temperature, Seeram et al. (2001)
investigated their stability in cell culture media at
physiological conditions. The degradation products of the
glycosides and the aglycone, cyanidin, identified in cell
29

culture media were benzoic acid derivatives: protocatechuic
acid, 2,4-dihydroxybenzoic acid, and 2,4,6trihydroxybenzoic acid (Figure 2).

30

Figure 2. Chemical Structures of Anthocyanin Metabolites

1

2

3
1

Protocatechuic acid

2

2,4-dihydroxybenzoic acid

3

2,4,6-trihydroxybenzoic acid

31

12. Plant Phenolics in Animal Studies
Animal studies investigating the effects of flavonoids
on tumor promotion have shown promising results. Issa et
al. (2007) demonstrated that green tea inhibited new
colonic adenomas in Apc

Min/+

mice injected with AOM;

however, it was not effective against larger tumors.
Previously, Kang et al. (2003) demonstrated that Apc

Min/+

mice that consumed diets containing anthocyanins, cyanidin,
or tart cherries had fewer cecal adenomas than mice that
consumed the control diet or sulindac. However, colonic
adenomas were not found to be significantly different among
treatment groups. Additionally, Bobe et al. (2006)
demonstrated that anthocyanins, when fed with sulindac to
Apc

Min/+

mice, were more effective in reducing small

intestinal tumors than sulindac alone. Similarly, Hagiwara
et al. (2001) demonstrated that purple corn anthocyanins
inhibited colorectal carcinogenesis in rats treated with
DMH and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine
(PhIP).
In a study investigating the impact of anthocyanins on
obesity, inflammation, and hyperlipidemia, which are
components of metabolic syndrome, researchers demonstrated
a significant link between cherry consumption and reduced

32

inflammatory markers. For 90 days, Zucker fatty rats were
fed a high fat diet with or without tart cherry powder.
Rats fed diets with cherry powder had reduced fat mass,
retroperitoneal TNF­‐α mRNA, plasma TNF­‐α, and decreased NF­‐
kB activity. The researchers also noted there was an
overall reduction of systemic and local inflammation
(Seymour et al., 2009).
An anthocyanin mixture from bilberries, and isolated
cyanidin-3-glucoside (C3G), were fed to Apc

Min/+

mice at

0.03, 0.01 or 0.3% in a standard AIN-93G diet for twelve
weeks. The researchers observed that the anthocyanin
mixture and the isolated C3G reduced colon tumors in a
dose-dependent manner when compared to mice consuming the
control diet. At the highest dose, tumor burden was
decreased 30 percent by the anthocyanin mixture, and 45
percent by C3G (Cooke et al., 2006).
In a murine experiment investigating the potential of
tart cherry anthocyanins to suppress inflammation-induced
behavior in rats, researchers demonstrated that
anthocyanins, in a dose-dependent manner, reduced pain
induced by heat, mechanical stimulation and edema (Tall et
al., 2004).
Finally, in a study using Wistar rats, cyanidin 3-O-βD-glycoside was shown to be rapidly absorbed and detected
33

in plasma. Its metabolites, protocatechuic acid and methyl
cyanidin 3-O-β-D-glycoside, were also detected in plasma and
tissue. The authors concluded these components may have the
potential to act as antioxidants in plasma and tissue
(Tsuda et al., 1999).

13. Apc
The Apc

Min/+
Min/+

Mouse Model and Azoxymethane
(multiple intestinal neoplasia) mouse model has

been extensively employed by this laboratory and others for
colorectal cancer research. These mice are highly
susceptible to intestinal adenoma formation, due to the Min
germline mutation, which is analogous to the human mutation
in FAP (Su et al., 1992). This similarity makes Apc

Min/+

mice a suitable model to study dietary interactions and
colorectal cancer, and to relate observations from animal
studies to human colorectal cancer. However, Apc

Min/+

mice

also commonly develop more than 30 adenomas throughout the
gastro-intestinal tract and have a life span of
approximately 120 days (Jackson Laboratory, 2013).
Azoxymethane is a potent colon-selective carcinogen
and stimulates colonic adenoma multiplicity (Issa et al.,
2007). The use of AOM increases the sensitivity of the

34

Apc

Min/+

mouse model for detecting effects on colonic

adenoma development (Mollersen et al., 2004).

35

RATIONALE AND SPECIFIC AIMS

Colorectal cancer is a major health problem in the
United States and the world. In the U.S., colorectal cancer
is the second most common cause of cancer mortality;
however, as much as 60 percent of colorectal cancers are
preventable. Diet can play a significant role in reducing
cancer risk. Previous research indicates eating a diet rich
in fruits and vegetables reduces the risk of developing
colorectal cancer. On the other hand, some dietary factors
increase colon cancer risk. For example, the relationship
between a high sucrose diet and the development of colonic
tumors or small-intestinal tumors has been demonstrated
epidemiologically and in previous research in this
laboratory and others.
Plant phenolics, such as anthocyanins, have been
demonstrated to have many health-promoting benefits.
Anthocyanins are natural red, purple, and blue flavonoid
pigments that impart color to fruits, vegetables, flowers
and plants. They have a wide variety of health­‐promoting
benefits attributed to their antioxidant and anti­‐
inflammatory properties. Research investigating the role of
anthocyanins for cancer prevention has shown promise.
However, much more information is needed to elucidate the

36

mechanisms whereby anthocyanins influence cancer risk. This
research aims to further understand the impacts of
anthocyanin consumption on intestinal tumor development in
animal models to relate to human colon cancer. This
research specifically investigated the efficacy of
anthocyanins to reduce colorectal tumorigenesis associated
with diets rich in sucrose and other dietary carbohydrates,
which have been shown to stimulate intestinal tumor
development.
The overall goal of this research was to determine the
effects of anthocyanin­‐rich tart cherry extracts on
intestinal adenoma development associated with feeding
carbohydrate-rich diets based on glucose, sucrose and starch
to Apc

Min/+

mice, a model of human colon cancer development.

The specific aims of this research were to:
1) Determine the efficacy of anthocyanin­‐rich
extracts to reduce intestinal adenoma development
in Apc

Min/+

mice consuming high­‐carbohydrate diets

(based on starch, sucrose and glucose), and
2) Relate changes in intestinal adenomas to
alterations in body weight and body composition.
The working hypothesis of specific aim one was that
anthocyanins will reduce intestinal adenoma development in

37

Apc

Min/+

mice compared to mice consuming control diets based

on dietary starch and diets not containing anthocyanins. We
further hypothesized that dietary sucrose and glucose will
increase intestinal adenoma development compared to dietary
starch, and that feeding anthocyanins will attenuate the
increase in tumorigenesis caused by these carbohydrate
sources. To test this hypothesis, the numbers and sizes of
intestinal adenomas was quantified at the end of a feeding
study wherein these carbohydrates were administered as the
sole carbohydrate sources in the diets fed to Apc

Min/+

mice

for nine weeks.
The working hypothesis of specific aim two was that diet
­‐induced differences in intestinal adenoma development will
be related to changes in body weight and body composition.
To test this hypothesis, body weights of mice were measured
weekly and total body fat and lean percentages were
measured at study termination. It was anticipated that mice
consuming diets rich in anthocyanins will have increased
body weight, lean body mass, and body fat compared to mice
not consuming anthocyanins.
At the conclusion of these studies, we expected to
have demonstrated that dietary anthocyanins reverse the
stimulatory effects of high­‐carbohydrate diets on intestinal

38

tumor development in Apc

Min/+

mice. These observations will

demonstrate the potential health benefits of anthocyanins
in human diets.

39

MATERIALS AND METHODS

1. Animals
An Apc

Min/+

mice breeding colony, originally purchased

from the Jackson Laboratory (Bar Harbor, ME), was
maintained in the University Research Containment Facility
at Michigan State University. This study was approved by
the Michigan State University All-University Committee on
Animal Use and Care. Mice were housed in a room with
constant humidity (40-50%), temperature (71-73°F), and a
12-hour day:night cycle. Mice used in this study were the
offspring produced when male C57BL/6J Apc

Min/+

mice were

mated with normal female C57BL/6J mice. Male Apc

Min/+

mice,

when not paired with normal female mice, where given
drinking water containing sulindac (200 mg/L) to reduce
morbidity caused by intestinal adenoma development. The
resulting offspring were randomly assigned to one of six
treatment groups at weaning, but were fed the control diet
from weaning until conclusion of the azoxymethane (AOM)
injection protocol (Figure 3). Mice were weaned between 21
and 28 days after birth and housed in plastic cages (1-4
mice/cage). After weaning, they received a series of
intraperitoneal AOM injections (8 mg/kg) once a week for
40

three weeks. After the three AOM injections, they were
given one week to recover before beginning their respective
experimental diets, which they were fed ad libitum for nine
weeks or until weight loss of 10 percent or greater was
detected. Approximately 13 males and 13 females were
assigned to each diet treatment. Genotype was determined
based on presence of small-intestinal adenomas observed at
the end of the study. At total of 159 mice were used in
this study. The numbers of female and male Apc

Min/+

mice

were 36 and 23, respectively. The numbers of female and
male wild type C57BL/6J mice were 49 and 51, respectively.
Mice were weighed weekly to track growth and monitor health
status. Six Apc

Min/+

mice and one wild type C57BL/6J mouse

were sacrificed early due to weight loss.

Figure 3. Experimental Design

41

2. Diets
Isocaloric experimental diets were based on the AIN93G diet (Reeves et al., 1993) and contained sucrose,
glucose, or starch as the sole carbohydrate source (Table
1). Fat content was increased from 7 to 15 percent to
reflect a typical human diet (approximately 31% of total
energy from fat). Micronutrients were increased to account
for the anticipated decreased food consumption caused by
the increased energy density of the diets. These diet
formulations were the three primary diets, of which each
contained 0 or 1500 parts per million (mg/kg) (PPM) of 50
percent pure anthocyanins from tart cherries. The
anthocyanins were supplied from the laboratory of Dr.
Muralee Nair, Michigan State University, and previously
used in experiments in this laboratory. The anthocyanins
used this project (Figure 4) were isolated from Montmorency
cherries and identified (Wang et al., 1997) as: 1) 3cyanidin 2”–O-β-D-glucopyranosyl-6”–O-α-L-rhamnopyranosyl-βD-glucopyranoside; 2) 3-cyanidin 6”–O-α-L-rhamnopyranosyl-βD-glucopyranoside; and 3) 3-cyanidin O-β-D-glucopyranoside
(Figure 4). Fresh and pitted cherries contained between
12.5-25.0 mg of anthocyanins per 100 g (Wang et al., 1999).
The composition of anthocyanins used in this experiment was

42

65 percent 3-cyanidin 2”–O-β-D-glucopyranosyl-6”–O-α-Lrhamnopyranosyl-β-D-glucopyranoside and 35 percent 3cyanidin 6”–O-α-L-rhamnopyranosyl-β-D-glucopyranoside (Kang
et al., 2003).

Figure 4. Chemical Structures of Anthocyanins from Tart
Cherries

Anthocyanin

R1

R2

3-cyanidin 2”–O-β-D-glucopyranosyl-6”–O-

Glucose

Rhamnose

H

Rhamnose

H

H

α-L-rhamnopyranosyl-β-D-glucopyranoside
3-cyanidin 6”–O-α-L-rhamnopyranosyl-β-Dglucopyranoside
3-cyanidin O-β-D-glucopyranoside

43

Table 1. Composition of Diets
Starch
(g/kg)
(control)
Casein (85% protein)
Corn Starch
Sucrose
Glucose
Soybean Oil
Cellulose
AIN-93G-MX
AIN-93-VX
L-Cysteine
Choline Bitartrate
Tert-Butylhydroquinone
+/- Anthocyanins

Glucose
(g/kg)

221
0
523
0
150
50
39
11
3
3
0.03
1.5

221
523
0
0
150
50
39
11
3
3
0.03
1.5

Sucrose
(g/kg)

221
0
0
523
150
50
39
11
3
3
0.03
1.5

3. Weight and Body Composition
Mice were weighed weekly starting from the day prior
to the first AOM injection until the day prior to
sacrifice. Body composition analysis was also performed the
day prior to sacrifice using the EchoMRI-100 body
composition analysis system for live animals (Echo Medical
Systems LLC, Houston, TX).

4. Sample Collection
Mice were euthanized after nine weeks of dietary
treatment by carbon dioxide asphyxiation. The terminal age
of mice was between 16-17 weeks. Blood samples were
immediately collected via cardiac puncture, transferred to

44

K2EDTA blood collection tubes, and centrifuged for 10
minutes at 1,300 (g). The plasma was transferred into fresh
microtubules and stored at -40°C for future analysis. Liver
and abdominal fat sections also were collected and stored
at -40°C for future analysis. The entire gastro-intestinal
tract from the small intestine to rectum was removed,
opened longitudinally and rinsed with cool water and
phosphate buffered saline (PBS). Individual sections (small
intestine, cecum, and colon) were pinned on cardboard and
fixed with 10 percent neutral buffered formalin overnight.
After fixation, a one-centimeter medial colonic section,
void of visible adenomas, was removed and preserved in
paraffin. The remaining fixed tissue segments were stained
with 0.3 percent methylene blue for three minutes, rinsed
with PBS, and stored in 1 percent neutral buffered
formalin.

5. Quantification of Intestinal Adenomas
Quantification of adenomas in the intestinal sections
was determined visually by using a Nikon SMZ
stereomicroscope by an observer blind to treatment. The
small intestine for each mouse was divided into three equal
sections (proximal, medial, and distal) at the time of

45

sacrifice. The colon was divided into two equal sections:
proximal and distal. Small intestinal adenomas were
quantified by total count and by two-dimensional size using
the formula: area = (π * d1 * d2)/4. Colonic and cecal
adenomas were quantified by number and three-dimensional
size using the following formula: volume = (π * d1 * d2 *
height)/6. The dimensions of the adenomas were measured to
the nearest 0.25 mm with a transparent grid placed under
the samples. Small intestinal adenomas are reported by
sections as: adenoma number, average size, and adenoma
burden (area). Colon tumors are reported as: adenoma
number, average size, adenoma burden (volume), adenomas per
tumor-bearing mouse, and adenoma incidence.

6. Statistical Analyses
Statistical analyses were performed using SAS software
(SAS Institute Inc., Cary, NC, Version 9.2). Body weights,
lean body mass percent, fat mass percent; and tumor number,
average adenoma size, and adenoma burden of each intestinal
segment were analyzed using analysis of variance (ANOVA)
with a 2 x 3 factorial arrangement, which represented two
anthocyanin concentrations x three dietary carbohydrates.
Parameters used in the statistical analysis included sex,

46

Apc status, anthocyanin, carbohydrate, and the interactions
between these parameters. When significant main effects
were detected, appropriate means were compared by the least
significant difference procedure. Statistical differences
were detected using a critical value of P<0.05 with the F
statistic. Results in tables are presented as least-square
means ± standard error of the mean (SEM). Colonic adenoma
incidence was analyzed with Fisher’s exact test using the
SAS frequency procedure.

47

RESULTS

1. Body Weights and Body Composition
Weekly average body weights of wild type C57BL/6J mice
and Apc

Min/+

mice, averaged across all dietary treatments,

are presented in Figure 5. Apc

Min/+

mice weighed less than

wild type C57BL/6J mice throughout the feeding period, with
significant differences (P<0.05) observed in every week
except weeks three and five. Final body weights of pooled
wild type C57BL/6J mice and Apc

Min/+

mice by sex are

presented in Figure 6. Final bodyweights of mice were
influenced by sex (Figure 6), and Apc gene status (Figures
7 and 8) (P<0.05).
Sex did not significantly influence mouse weight at
the start of the experiment. However, beginning with the
second week of dietary treatment, male mice weighed
significantly more than female mice. This trend continued
for the duration of the experiment. At termination of this
study, male Apc

Min/+

mice weighed more than their female

counterparts (Figure 7), with terminal weights of 24.30 ±
0.86 and 20.64 ± 0.76 g, respectively.

48

Weekly average body weights of pooled wild type
C57BL/6J mice and Apc

Min/+

mice by carbohydrate source are

presented in Figure 9. Carbohydrate source did not have a
significant effect on body weights of pooled mice at the
conclusion of the study; however significant (P<0.05)
differences were detected in weeks seven, eight and ten.
During these weeks, mice consuming sucrose weighed more
than mice consuming starch or glucose.
Weekly average body weights of female and male Apc

Min/+

mice consuming different diets are presented in Figures 10
and 11. No significant sex x carbohydrate x anthocyanin
interactions were detected for body weight during the
duration of the experiment.
Terminal body fat and lean body mass percentages are
presented in Figures 12 and 13. Apc status was the only
factor that significantly (P<0.05) influenced these
parameters. Wild type C57BL/6J mice had significantly more
body fat and less lean mass on a percentage basis than
Apc

Min/+

mice.

2. Small Intestinal Adenomas
Adenoma number, average size, and burden were assessed
in the proximal, medial, and distal segments of the small

49

intestine. Values for each segment were summed to obtain a
composite value for the whole small intestine and these
data are presented in Table 2. Total small intestinal
adenoma number was significantly influenced by sex
(P<0.05). Female mice (51.68 ± 4.02) had a significantly
greater number of small intestinal adenomas than males
(38.67 ± 5.03). However, mean adenoma size and total
adenoma burden were not significantly influenced by sex. A
sex x carbohydrate x anthocyanin interaction was detected
for total small intestinal adenoma number and burden
(P<0.05), but not average adenoma size. Females consuming
glucose had the greatest number of adenomas and burden
compared to all groups. Female mice that consumed
anthocyanin-glucose had significantly fewer adenomas (56.50
± 11.42) and significantly less adenoma burden (84.12 ±
2

25.13 mm ) than females consuming glucose (105.60 ± 10.21)
2

and (196.51 ± 22.47 mm ), respectively. Males consuming
glucose had the fewest adenomas and lowest burden, but this
was not significantly different from males that consumed
the control or anthocyanin-glucose diets. No other
differences within carbohydrate sources were detected.
Proximal small intestinal data are presented in Table
3. Adenoma number in females (11.92 ± 0.82) was

50

significantly (P<0.05) greater in the proximal small
intestine than in males (7.97 ± 1.06), but a significant
difference for average size or burden was not detected
based on sex. Proximal small intestinal burden was
significantly (P<0.05) influenced by a sex x carbohydrate x
anthocyanin interaction, but adenoma number and average
size were not. Female mice consuming glucose had the
2

greatest adenoma burden (50.96 ± 7.54 mm ), which was
significantly (P<0.05) different from females consuming
2

anthocyanin-glucose (27.49 ± 8.43 mm ) and sucrose (25.92 ±
5.96). Males consuming glucose had the lowest proximal
2

small intestinal adenoma burden (6.49 ± 6.89 mm ) of all
mice, which was significantly (P<0.05) different than males
consuming anthocyanin-glucose, sucrose, and anthocyaninsucrose. Average adenoma burden in males consuming glucose
was also significantly different (P<0.05) from all female
diet groups except those that consumed anthocyanin-glucose.
Medial small intestinal adenoma data are presented in
Table 4. Medial small intestinal adenoma number and burden
tended (P<0.10) to be influenced by a sex x carbohydrate x
anthocyanin interaction, but average size was not. No
significant differences were detected for average adenoma

51

number, average adenoma size, or burden for any interaction
analyzed.
Distal small intestinal adenoma data are presented in
Table 5. No significant differences were associated with
sex. However, distal small intestinal adenoma number tended
(P<0.10) to be influenced by a sex x carbohydrate x
anthocyanin interaction. Additionally, a sex x carbohydrate
x anthocyanin interaction was detected for mean burden
(P<0.05). Female mice consuming glucose showed the greatest
2

adenoma burden (80.27 ± 10.87 mm ) compared to all treatment
groups for both sexes (P<0.05). Female mice consuming
2

sucrose had the lowest adenoma burden (6.33 ± 8.59 mm ), but
this difference was only observed to be different when
compared to females consuming glucose and to males
consuming cornstarch or anthocyanin-sucrose.

3. Colonic and Cecal Adenomas
Colonic adenoma number, incidence, average size, and
burden are presented in Tables 6, 7, and 8. No cecal
adenomas were observed in any mouse in this study. Colonic
adenomas were only observed in Apc

Min/+

mice.

Adenoma number and incidence are presented in Table 6.

52

Adenoma number and incidence were not significantly
influenced by sex or dietary treatment.
Average adenoma size and burden for all Apc

Min/+

mice

are presented in Table 7. Sex did not influence average
size or burden; however, an anthocyanin x carbohydrate
interaction significantly (P<0.05) influenced mean colonic
adenoma size in all Apc

Min/+

mice. Mice that consumed
3

sucrose had significantly larger adenomas (16.41 ± 3.06 mm )
than all mice except those that consumed anthocyanin3

cornstarch (8.67 ± 3.06 mm ).
Average adenoma size and burden for adenoma-bearing
Apc

Min/+

mice are presented in Table 8. Sex did not

influence average size or burden; however, an anthocyanin x
carbohydrate interaction significantly (P<0.05) influenced
average colonic adenoma size in adenoma bearing Apc

Min/+

mice. Mice that consumed sucrose had significantly (P<0.05)
3

larger adenomas (24.94 ± 3.61 mm ) than all mice. Mice that
consumed anthocyanin-sucrose had the smallest average
3

adenoma size (6.05 ± 3.74 mm ), but this was only
significantly different from mice that consumed sucrose
(P<0.05). Mice that consumed anthocyanin-sucrose had

53

adenomas that were 75.8 percent smaller compared to mice
only consuming sucrose. Adenoma burden among adenomabearing mice was not influenced by dietary treatment.

54

Figure 5. Weekly average body weights of wild type C57BL/6J
Min/+
and Apc
mice.

30!

Average weight (grams)!

25!
*	
  

20!

*	
  

15!
ApcMin/+ Mice
Wild type Mice

10!

5!

0!
0!

*

1!

2!

3!

4!

5!

6! 7! 8!
Weeks!

9! 10! 11! 12! 13!

Denotes weeks without significant difference (P>0.05).

55

Figure 6. Final body weights of pooled male and female wild
Min/+
type C57BL/6J and Apc
mice.

b

30

Weight (grams)

25

a

20

15

10

5

0
Females

Males
Sex

ab

Means not sharing a common superscript are different (P<0.05).

56

Figure 7. Final body weights of male and females by wild
Min/+
type C57BL/6J and Apc
status.
c

35

30

Weight (grams)

25

b

b
a

20

15

10

5

0
ApcMin/+
Females

ApcMin/+ Wild type Wild type
Males
Females
Males
Apc Status and Sex

abc

Means not sharing a common superscript are different
(P<0.05).

57

Figure 8. Weekly average body weights of male and female
Min/+
wild type C57BL/6J and Apc
mice.

40

35

Average weight (grams)

30

25

20

15
ApcMin/+ Female
Wild type Female

10

ApcMin/+ Male
Wild type Male
5

0
0

1

2

3

4

5

6

7
8
Weeks

58

9

10

11

12

13

14

Figure 9. Carbohydrate effect on pooled C57BL/6J mice and
Min/+
Apc
mice weekly mean body weights.
30

28
*

26
*
*

24

Average weight (grams)

22

20
Starch
Sucrose
Glucose

18

16

14

12

10
0

1

2

3

4

5

6
7
Weeks

*

8

9

10

11

12

13

Denotes significant difference between sucrose fed mice and
mice fed starch or glucose (P<0.05).

59

Min/+

Figure 10. Weekly average female Apc
mice body weights
when fed diets without (-) or with (+) 1500 ppm
anthocyanins: (-) cornstarch, (-) sucrose, (-) glucose, (+)
cornstarch, (+) sucrose, or (+) glucose.

24

22

Average weight (grams)!

20

18

Starch

16

Sucrose

14
Glucose
Anthocyanin-Starch

12

Anthocyanin-Sucrose
10

Anthocyanin-Glucose

8
0

1

2

3

4

5

6

7 8
Weeks!

60

9

10 11 12 13 14

Min/+

Figure 11. Weekly average male Apc
mice body weights
when fed diets without (-) or with (+) 1500 ppm
anthocyanins: (-) cornstarch, (-) sucrose, (-) glucose, (+)
cornstarch, (+) sucrose, or (+) glucose.
30

!

28

Average weight (grams)!

26

24

22

20
Starch
18

Sucrose
Glucose

16

Anthocyanin-Starch

14

Anthocyanin-Sucrose
12
Anthocyanin-Glucose
10
0

1

2

3

4

5

6

7 8 9
Weeks !

61

10 11 12 13 14

Figure 12. Mean lean body mass percentage of wild type
Min/+
C57BL/6J and Apc
mice.
25

a	
  

20
Percent

	
  	
  	
  	
  	
  b	
  

15
10
5
0
Wild type
ApcMin/+
Apc Gene Status

ab

Means not sharing a common superscript are different (P<0.05).

Percent

Figure 13. Mean lean body mass percentage of wild type
Min/+
C57BL/6J and Apc
mice.
82
80
78
76
74
72
70
68
66
64

	
  	
  	
  	
  	
  b	
  

	
  	
  	
  	
  	
  a	
  

Wild type
ApcMin/+
Apc Gene Status
ab

Means not sharing a common superscript are different (P<0.05).

62

Table 2. Total small intestine adenoma number, average
Min/+
size, and burden in Apc
mice fed diets without (-) or
with (+) 1500 ppm anthocyanins: (-) cornstarch, (-)
sucrose, (-) glucose, (+) cornstarch, (+) sucrose, or (+)
glucose.

#

TOTAL SMALL

Adenoma

INTESTINE

Number

Sex

Average Size
2

(mm )

Total Burden

*

2

(mm )

+

Female
Male

a

51.68 ± 4.02
b
38.67 ± 5.03

1.96 ± 0.13
1.91 ± 0.16

96.36 ± 8.85
79.81 ± 11.08

Treatment (Female)
bcd
(-) Cornstarch 35.00 ± 11.42

2.26 ± 0.37

78.60 ± 25.13

(-) Sucrose
(-) Glucose

2.06 ± 0.26
1.90 ± 0.31

46.71 ± 17.77
a
196.51 ± 22.47

(+) Cornstarch 40.25 ± 8.08
bc
(+) Sucrose
48.83 ± 9.32

2.38 ± 0.26
1.73 ± 0.30

90.84 ± 17.77
bc
81.36 ± 20.52

(+) Glucose
56.50 ± 11.42
Treatment (Male)
bcd
(-) Cornstarch 41.67 ± 13.19
bcd
(-) Sucrose
34.67 ± 13.19

1.46 ± 0.37

84.12 ± 25.13

2.02 ± 0.42
2.51 ± 0.42

94.47 ± 29.01
bc
86.98 ± 29.01

(-) Glucose
23.00 ± 9.32
bcd
(+) Cornstarch 39.33 ± 13.19

1.15 ± 0.30
1.80 ± 0.42

30.44 ± 20.52
bc
75.31 ± 29.01

(+) Sucrose
(+) Glucose

1.91 ± 0.37
2.09 ± 0.42

97.86 ± 25.13
bc
93.82 ± 29.01

cd

23.88 ± 8.08
a
105.60 ± 10.21
bcd
b

c

#

*

b

53.00 ± 11.42
bcd
40.33 ± 13.19

bc

bc
b

bc
bc
c
b

Data presented as least square mean ± SEM.

Total burden equal to the sum of all adenomas within the
intestinal segment.
+
Sex is considered a different category from sex x treatment.
abcd
Means not sharing a common superscript within a column and
category are different (P<0.05).

63

Table 3. Proximal small intestine adenoma number, average
Min/+
size, and burden in Apc
mice fed diets without (-) or
with (+) 1500 ppm anthocyanins: (-) cornstarch, (-)
sucrose, (-) glucose, (+) cornstarch, (+) sucrose, or (+)
glucose.

#

PROXIMAL SMALL

Adenoma

INTESTINE

Number

Sex

Average Size
2

(mm )

Total Burden

*

2

(mm )

+

Female
11.92 ± 0.82
b
Male
7.97 ± 1.06
Treatment (Female)
(-) Cornstarch 8.40 ± 2.14

a

3.28 ± 0.31
3.22 ± 0.40

34.25 ± 2.90
26.28 ± 3.72

4.20 ± 0.81

31.38 ± 7.54

7.13 ± 1.69
20.80 ± 2.14

3.70 ± 0.64
2.78 ± 0.81

25.92 ± 5.96
a
50.96 ± 7.54

(+) Cornstarch 8.75 ± 1.69
(+) Sucrose
12.67 ± 1.96

4.43 ± 0.64
2.69 ± 0.74

36.51 ± 5.96
ab
33.26 ± 6.89

(+) Glucose
13.75 ± 2.40
Treatment (Male)
(-) Cornstarch 5.67 ± 2.77

1.93 ± 0.91

27.49 ± 8.43

4.89 ± 1.05

30.30 ± 9.74

(-) Sucrose
(-) Glucose

10.00 ± 2.77
4.83 ± 1.97

3.58 ± 1.05
1.60 ± 0.74

34.23 ± 9.74
c
6.49 ± 6.89

(+) Cornstarch 6.33 ± 2.77
(+) Sucrose
12.75 ± 2.40

3.52 ± 1.05
2.56 ± 0.91

22.01 ± 9.74
ab
29.13 ± 8.43

(+) Glucose

3.16 ± 1.05

35.51 ± 9.74

(-) Sucrose
(-) Glucose

#

*

8.33 ± 2.77

ab
b
ab
bc
abc
ab
bc
ab

Data presented as least square mean ± SEM.

Total burden equal to the sum of all adenomas within the
intestinal segment.
+
Sex is considered a different category from sex x treatment.
abc
Means not sharing a common superscript within a column and
category are different (P<0.05).

64

Table 4. Medial small intestine adenoma number, average
Min/+
size, and burden in Apc
mice fed diets without (-) or
with (+) 1500 ppm anthocyanins: (-) cornstarch, (-)
sucrose, (-) glucose, (+) cornstarch, (+) sucrose, or (+)
glucose.

#

MEDIAL SMALL

Adenoma

INTESTINE

Number

Sex
Female
18.58 ± 1.76
Male
13.88 ± 2.25
Treatment (Female)
(-) Cornstarch 13.00 ± 4.56
(-) Sucrose
8.25 ± 3.60
(-) Glucose
36.20 ± 4.56
(+) Cornstarch 15.25 ± 3.62
(+) Sucrose
18.50 ± 4.17
(+) Glucose
20.25 ± 5.10
Treatment (Male)
(-) Cornstarch 16.33 ± 5.89
(-) Sucrose
10.33 ± 5.89
(-) Glucose
10.00 ± 4.17
(+) Cornstarch 11.67 ± 5.89
(+) Sucrose
17.25 ± 5.10
(+) Glucose
17.67 ± 5.89
#

*

Average Size
2

(mm )

Total Burden

*

2

(mm )

2.04 ± 0.18
1.75 ± 0.23

35.51 ± 3.51
26.02 ± 4.50

2.28
1.74
1.85
2.60
1.96
1.79

±
±
±
±
±
±

0.47
0.37
0.47
0.37
0.43
0.52

30.25
14.46
65.27
35.56
30.21
37.31

±
±
±
±
±
±

9.12
7.21
9.12
7.21
8.33
10.20

1.28
2.46
1.27
1.63
1.76
2.07

±
±
±
±
±
±

0.61
0.61
0.43
0.61
0.52
0.61

24.34
26.77
15.41
23.30
29.89
36.39

±
±
±
±
±
±

11.77
11.77
8.33
11.77
10.20
11.77

Data presented as least square mean ± SEM.

Total burden equal to the sum of all adenomas within the
intestinal segment.
Means in this table are not different (P>0.05).

65

Table 5. Distal small intestine adenoma number, average
Min/+
size, and burden in Apc
mice fed diets without (-) or
with (+) 1500 ppm anthocyanins: (-) cornstarch, (-)
sucrose, (-) glucose, (+) cornstarch, (+) sucrose, or (+)
glucose.

#

DISTAL SMALL

Adenoma

INTESTINE

Average Size

Number

2

(mm )

Total Burden

*

2

(mm )

+

Sex
Female
21.50 ± 1.97
Male
16.81 ± 2.47
Treatment (Female)
(-) Cornstarch 15.50 ± 5.60
(-) Sucrose
8.50 ± 3.96

1.20 ± 0.15
1.35 ± 0.18

27.36 ± 4.28
27.52 ± 5.36

1.33 ± 0.41
1.20 ± 0.30

21.56 ± 12.15
c
6.33 ± 8.59

(-) Glucose
48.60 ± 5.01
(+) Cornstarch 16.25 ± 3.96

1.58 ± 0.38
1.40 ± 0.30

80.27 ± 10.87
bc
18.77 ± 8.59

(+) Sucrose
17.67 ± 4.57
(+) Glucose
22.50 ± 5.60
Treatment (Male)
(-) Cornstarch 19.67 ± 6.47

0.87 ± 0.34
0.80 ± 0.42

17.89 ± 9.92
bc
19.31 ± 12.15

1.53 ± 0.48

39.83 ± 14.03

(-) Sucrose
(-) Glucose

14.33 ± 6.47
8.17 ± 4.57

1.82 ± 0.48
0.84 ± 0.34

25.98 ± 14.03
bc
8.54 ± 9.92

(+) Cornstarch 21.33 ± 6.47
(+) Sucrose
23.00 ± 5.60

1.15 ± 0.48
1.31 ± 0.42

29.99 ± 14.03
b
38.83 ± 12.15

(+) Glucose

1.44 ± 0.48

21.93 ± 14.03

#

*

14.33 ± 6.47

bc
a

bc

b
bc
bc
bc

Data presented as least square mean ± SEM.

Total burden equal to the sum of all adenomas within the
intestinal segment.
+
Sex is considered a different category from sex x treatment.
ab

Means not sharing a common superscript within a column and
category are different (P<0.05).

66

Table 6. Colon adenoma number per mouse, adenoma number per
Min/+
adenoma bearing mouse, and percent incidence in Apc
mice fed diets without (-) or with (+) 1500 ppm
anthocyanins: (-) cornstarch, (-) sucrose, (-) glucose, (+)
cornstarch, (+) sucrose, or (+) glucose.
COLON

#

Adenoma Number/Mouse Adenoma Number/Tumor
Bearing Mouse

Sex
Female
Male
Treatment
(-) Cornstarch
(-) Sucrose
(-) Glucose
(+) Cornstarch
(+) Sucrose
(+) Glucose

Incidence
(%)

1.67 ± 0.41
0.89 ± 0.53

2.63 ± 0.55
1.42 ± 0.80

63.8 ± 8.12
57.1 ± 9.52

0.47
2.52
1.05
1.06
1.66
0.92

1.00
4.00
1.75
1.39
2.50
1.50

50.0
63.6
54.5
81.8
60.0
42.9

±
±
±
±
±
±

0.87
0.81
0.72
0.81
0.77
0.91

#

±
±
±
±
±
±

1.42
1.03
1.07
0.99
1.07
1.42

Data presented as least square mean ± SEM.
Means in this table are not different (P>0.05).

67

±
±
±
±
±
±

18.9
15.2
15.7
12.2
16.3
20.2

Table 7. Average colon adenoma size and adenoma burden in
Min/+
all Apc
mice fed diets without (-) or with (+) 1500 ppm
anthocyanins: (-) cornstarch, (-) sucrose, (-) glucose, (+)
cornstarch, (+) sucrose, or (+) glucose.
COLON

Average Adenoma Size
3

#

Adenoma Burden

(mm )
Sex
Female
Male
Treatment
(-) Cornstarch

*

3

(mm )

5.82 ± 1.55
7.78 ± 1.99
4.68 ± 3.29

13.26 ± 3.52
9.71 ± 4.51
b
a

4.68 ± 7.47

(-) Sucrose
(-) Glucose

16.41 ± 3.06
b
3.15 ± 2.73

(+) Cornstarch
(+) Sucrose

8.67 ± 3.06
b
3.80 ± 2.91

10.06 ± 6.92
9.76 ± 6.60

(+) Glucose

4.11 ± 3.45

9.64 ± 7.81

#

*

ab
b

29.79 ± 6.92
4.96 ± 6.19

Data presented as least square mean ± SEM.

Total burden equal to the sum of all adenomas within the
intestinal segment.
ab
Means not sharing a common superscript within a column and
category are different (P<0.05).

68

Table 8. Average colon adenoma size and adenoma burden in
Min/+
adenoma bearing Apc
mice fed diets without (-) or with
(+) 1500 ppm anthocyanins: (-) cornstarch, (-) sucrose, (-)
glucose, (+) cornstarch, (+) sucrose, or (+) glucose.
COLON

Average Adenoma Size
3

Adenoma Burden
(mm )

Sex
Female
8.80 ± 1.91
Male
13.07 ± 2.79
Treatment
b
(-) Cornstarch 8.85 ± 4.98
24.94 ± 3.61
b
7.18 ± 3.74

(+) Cornstarch 11.31 ± 3.46
b
(+) Sucrose
6.05 ± 3.74
(+) Glucose
#

7.22 4.98

*

3

(mm )

(-) Sucrose
(-) Glucose

#

20.65 ± 4.79
15.55 ± 7.00
8.85 ± 12.52

a

46.35 ± 9.07
10.63 ± 9.39

b

13.12 ± 8.69
15.05 ± 9.39

b

14.60 ±12.52

Data presented as least square mean ± SEM.

*

Total burden equal to the sum of all adenomas within the
intestinal segment.
ab
Means not sharing a common superscript within a column and
category are different (P<0.05).

69

DISCUSSION

Apc

Min/+

mice weighed less than wild type C57BL/6J mice

throughout the feeding period, with significant differences
observed in every week except weeks three and five (Figure
5); however, body weights of both groups were significantly
different (P<0.05) at conclusion of this study. Body weight
differences also were observed to be influenced by a
significant sex x Apc status interaction, with Apc

Min/+

males weighing less than wild type C57BL/6J males, and
Apc

Min/+

females weighing less than wild type C57BL/6J

females at the conclusion of this study. At termination,
Apc
Apc

Min/+
Min/+

males weighed approximately four grams more than
females (Figures 7 and 8). This observation is

consistent with previous findings in our laboratory using
several dietary interventions. The weight loss observed in
Apc

Min/+

mice is highly correlated to morbidity associated

with adenoma development in Apc

Min/+

mice. Weight loss

increases as the average size of small intestinal tumors
increases. When adenomas are approximately 1.5 mm in
diameter or greater, adenomas have the tendency to
hemorrhage and increase the risk of morbidity (Kang, 2005).
70

These observations also are consistent with a study
conducted by The Jackson Laboratory (JAX) where mean
weights of male and female wild type C57BL/6J mice were
29.71 and 21.99 g at 16 weeks of age (Jackson Laboratory,
2013). The mice in this study were also 16-17 weeks of age
at termination, and body weights of wild type C57BL/6J male
and female mice were 30.79 and 24.38 g respectively,
suggesting AOM treatment did not influence body weight in
wild type C57BL/6J mice
Body weights of Apc

Min/+

mice were less than wild type

C57BL/6J mice in this study; however, it cannot be
determined if this can be specifically attributed to
tumorigenesis associated to the Apc knockout or AOM
treatment, or a combination of both factors. However, these
observations do suggest that tumorigenesis begins as early
as three to four weeks of life and influences body weight
early in development.
Dietary treatment did not have a significant influence
on body weights of Apc

Min/+

mice at the conclusion of the

study. Previous studies in this laboratory demonstrated
mice fed sucrose weighed significantly more than mice fed
starch (Wang, 2005; Powell, 2011). Although similar trends

71

were observed in this study, they were not statistically
significant.
Mouse body fat and lean mass were only influenced by
Apc status in this study, with Apc

Min/+

mice having less

body fat and higher lean mass compared to their wild type
counterparts on a percentage basis. This was a similar
trend observed by Powell (2011); however, Powell (2011)
also observed significant differences associated with
similar dietary treatments based on starch, fructose,
glucose, and sucrose.
In this study, Apc status and/or sex were the only
factors that significantly influenced weight and body
composition. These results contrast with previous findings
from this laboratory, which can be attributed to the use of
an aggressive treatment, AOM treatment in an Apc

Min/+

mouse

model, and a longer dietary treatment period compared to
Powell (2011). Although mice tolerated AOM injections well,
AOM injections may have contributed to weight loss, and
mice that lost weight tended to lose weight during the
final three weeks and this may have contributed to results
with non-significant differences.
The number of total small intestinal adenomas was
significantly greater in females than males. This

72

difference was also observed in the proximal section of the
small intestine, but not the medial or distal sections.
Average adenoma size and burden were not different between
sexes. The net effect of this observation is sex influenced
tumor multiplicity. This observation is consistent with
research results from this laboratory using the same mouse
model (Powell, 2011; Kang, 2002; Cao, 2012).
In this study, a sex x carbohydrate x anthocyanin
interaction was detected for total small intestinal adenoma
number and adenoma burden. Female mice consuming sucrose
had the second fewest total small intestinal adenomas among
all treatment groups. However, females that consumed
anthocyanin-sucrose did not show a significantly different
number of adenomas, average size or burden from those that
consumed only sucrose. Distal small intestinal adenoma
burden was the lowest in females consuming sucrose, which
can likely be the reason females had second lowest total
small intestinal burden of all treatment groups. Distal
small intestinal adenoma burden was lower than what was
observed in the proximal and medial small intestine of
females. This may be attributed to sucrose’s components, Dglucose and fructose, primarily being absorbed in the
proximal and medial sections (Wright, 1993; Douard and
Ferraris, 2008). Additionally, sucrose has been shown to

73

promote intestinal proliferation in murine models (Wang,
2005; Caderni et al., 1991; Caderni et al., 1997). This may
explain why female mice consuming sucrose had a greater
burden in the upper two-thirds of the small intestine
compared to the distal third, and why total small
intestinal burden was lower. However, this observation was
only significantly different for females that consumed
glucose.
Female mice that consumed glucose showed the greatest
total small intestinal adenoma number among all groups.
Many types of cancer cells have increased glucose uptake
(Taubes, 2012). This observation may be explained by the
Warburg effect, as described by Otto Warburg in the 1920s.
He observed that cancer cells utilize glucose in a process
called aerobic glycolysis. In this process, cancer cells
convert glucose -> pyruvate -> lactate, which is used as a
primary energy source in cells’ cytoplasm (Taubes, 2012).
Researchers have shown the enzyme M2-pyruvate kinase
(M2PK), which converts phosphoenolpyrutave to pyruvate, can
be used as a biomarker for colorectal cancer because it is
highly expressed in cancer cells (Kumar et al, 2007; Hardt
et al, 2004). Reducing the activity of this enzyme can
reverse the Warburg effect and thus implicates (M2PK) as a
key component of glucose metabolism in cancer cells

74

(Christofk et al., 2008). The Warburg effect also may
explain why females consuming glucose had the greatest
burden in the distal small intestine (Table 5). Since
cancer cells have been shown to prefer glucose as an energy
source, it is possible adenomas in the distal small
intestine had increased glucose utilization in this study,
which may lead to an increased capacity for proliferation.
Dietary polyphenols have been shown in vitro to
decrease glucose uptake in human cancer cell lines and in
animal cells (Kobayashi et al., 2000; Johnston et al.,
2005). In Caco-2 cancer cells, activity of sodium-dependent
glucose transporters was inhibited by flavonoid glycosides
and naturally occurring dietary aglycones. However,
aglycones (those that are produced by hydrolysis during
digestion) and phenolic acids did not inhibit glucose
transportation. In sodium-free conditions, aglycones
inhibited glucose transportation, whereas the glycosides
and phenolic acids did not. These results suggest aglycones
may inhibit glucose transporters like those in the GLUT
family, while the glycosides may inhibit sodium-dependent
active transporters (Johnston, 2005). In rat tissue, green
tea (-)-epicatechin gallate and (-)-epigallocatechin
gallate has been shown to inhibit glucose transport by
acting as a sodium-dependent glucose transporter 1 (SLGT1)

75

antagonist in vitro (Kobayashi et al., 2000). Additionally,
the glucose transporter (GLUT2), which is found on both the
apical and basolateral membrane of the small intestines
(Wright, 1993), has been demonstrated to transport
anthocyanins in vitro (Kobayashi et al., 2000). In Caco-2
cells, treatment with anthocyanins decreased glucose
transport while increasing GLUT2 expression (Faria et al.,
2009).
In this study, anthocyanins consumption was associated
with a decrease in tumorigenesis in females consuming
glucose, with a site-specific influence in the proximal and
distal small intestine. No other relationships between diet
and adenoma development for female mice can be established
based on the results of this study.
The results for male mice are less defined. There were
no significant differences in adenoma number when comparing
mice consuming cornstarch, sucrose, or glucose to their
corresponding anthocyanin containing diets in the whole
small intestine or individual intestinal sections. Adenoma
burden in male mice was not influenced by diet in the whole
small intestine; however, adenoma burden in the proximal
small intestine was significantly greater for males
consuming anthocyanin-glucose compared to glucose. This
observation is the opposite of what was observed in female

76

mice. There may be two reasons for this difference.
Firstly, three male mice consuming glucose were sacrificed
five weeks early due to a weight loss of 10 percent.
Secondly, only four male mice completed the dietary
treatment for this group. These factors contributed to an
incomplete data set for this treatment group.
The differences between sexes for mice consuming
glucose with or without anthocyanins are too inconsistent
to formulate a hypothesis for the difference observed
between sexes in this study.
Additionally, it has been demonstrated anthocyanins
and anthocyanidins act as antioxidants and antiinflammatory agents (Seeram et al., 2001; Seeram et al.,
2003, Mulabagal et al., 2009), which may explain how
anthocyanins in this study reduced tumor number and burden
in female mice consuming glucose.
Anthocyanins have been shown to be inhibitors of COX
activity and inflammation. Since inflammation has not been
strongly linked to sporadic colorectal cancer, it is
unlikely to be a significant initiator of colorectal cancer
(Terzic et al., 2010). However, chronic inflammation and
up-regulation of COX-2 and increased levels of its
prostaglandin products have been linked to tumor promotion
(Taketo, 1998; Yang et al., 1998). Furthermore, NSAIDs such

77

as sulindac and Licofelone ([2,2-dimethyl-6-(4-chloropheny7-phenyl-2,3-dihydro-1H-pyrrazoline-5-yl] acetic acid) have
been shown experimentally to reduce small intestinal
adenoma number in the same mouse model used in this study
(Kang, 2005; Mohammed et al, 2011). The combination of
anthocyanins and sulindac also has been shown to reduce
tumor number and total adenoma burden, but not average size
(Bobe et al., 2006). This combination may be more effective
at reducing COX activity than anthocyanins alone, which was
also observed by Wang et al. (1999).
In this study, it is hypothesized that anthocyanins
are absorbed in the small intestine by GLUT2 and inhibit
SGLT1 and GLUT2 glucose absorption (Faria et al., 2009;
Kobayashi et al., 2000; Johnston et al., 2005). Then,
anthocyanins, or anthocyanidins after removal of sugar
residues, may act as anti-inflammatory and antioxidant
agents (Tsuda et al., 1999; Seeram et al., 2003; Wang et
al., 1999); thereby reducing small intestinal adenoma
burden associated with glucose consumption.
In this study, carbohydrate source did not
significantly influence colon adenoma number, burden or
incidence. These results are consistent with those observed
by Wang (2005) in his first study and by Powell (2011).
Additionally, no cecal adenomas were observed in any mouse,

78

which is in contrast to the results observed by Kang
(2002). However, other researchers have also observed a low
number of cecal tumors in studies using Apc

Min/+

mice

(Erdman et al., 2005; and Yang et al., 2003).
Previous research in this laboratory has yielded
consistent results with colonic tumor multiplicity in diets
with varying carbohydrate sources. Mice that consumed
sucrose had the same number of adenomas compared to mice
consuming starch (Wang et al., 2009; Powell, 2011; and
Wang, 2005). The numbers of colonic adenomas in mice
consuming varying carbohydrates in this study was generally
greater than the number observed by Powell (2011) and Wang
(2005), which indicates the AOM injections may have
influenced tumor multiplicity as anticipated. This increase
in tumor multiplicity associated with AOM treatment is
consistent with findings by Suzui et al. (2002) and Issa et
al. (2007).
A significant carbohydrate x anthocyanin interaction
was observed for average colonic adenoma size (Tables 7 &
8). Mice consuming sucrose had significantly larger
adenomas compared to all other diets. Wang et al. (2009)
also observed that sucrose significantly influenced adenoma
incidence and size when compared to mice consuming starch.
However, Wang’s observation was not significant in adenoma79

bearing mice (Wang et al., 2009). It has been hypothesized
that sucrose consumption, in this model and other murine
models, promotes intestinal proliferation. Increased cell
proliferation by sucrose has been demonstrated with Ki67
labeling (Wang, 2005). Enhanced aberrant crypt formation
has also been demonstrated in rodents fed sucrose (Caderni
et al., 1991; Caderni et al., 1997). Short-term highsucrose consumption increases oxidative stress in rats
(Busserolles et al., 2002). Therefore, it is reasonable to
suggest that long-term consumption of high-sucrose diets
may have contributed to consistent oxidative stress in this
study thereby increasing the likelihood for additional
genetic mutations and adenoma promotion.
The link between sucrose consumption and elevated
inflammatory markers also was observed in a dietary
intervention study involving 41 overweight patients. The
study demonstrated that consumption of high sucrose diets
increased serum levels of haptoglobin and transferrin
(Sorensen et al., 2005). Similarly, epidemiological data
from the Nurses’ Health Study positively correlated sugar
and refined grain consumption with inflammatory markers
such as IL-6 and TNF-α (Schulze et al., 2005).

80

In this study, mice consuming the anthocyanin-sucrose
diet demonstrated a reduced colonic adenoma burden when
compared to the mice consuming only sucrose.
Anthocyanins can be absorbed as intact glycosides
(Miyazawa et al., 1999). Cyanidin 3-O-β-D-glycoside (C3G)
and one of its metabolites, protocatechuic acid, has also
been detected in the plasma of rats given C3G orally by
gavage (Tsuda et al., 1999). Additionally, methylated C3G
has been detected in liver and kidney tissue (Tsuda et al.,
1999). In a human study, anthocyanins were detected in
plasma and urine samples after test subjects consumed
cherry powder, and antioxidant capacity of plasma increased
after cherry ingestion (Kirakosyan et al., 2010). Finally,
Zucker rats fed cherry powder had decreased plasma
inflammatory markers (Seymour et al., 2009). These results
support the potential of anthocyanins to be absorbed and
act as antioxidants or anti-inflammatory agents in vivo.
The hypothesized mechanism by which anthocyanins
reduce colonic adenoma promotion is by inhibiting COX-I and
COX-II enzymatic activity and thereby reducing
inflammation, a hallmark of cancer. Animal studies
investigating this hypothesis have shown that consumption
of tart cherries reduces plasma TNF-α and IL-6, and mRNA

81

TNF-α and IL-6, which are key regulators of COX enzyme
expression (Seymour et al., 2009).
Additionally, cell culture studies investigating the
effect of various anthocyanins and anthocyanidins on COX
enzyme expression support this hypothesis (Seeram et al.,
2001; Seeram et al, 2003; Wang et al., 1999).

82

SUMMARY AND CONCLUSIONS

This study demonstrated that intestinal tumor
development in Apc

Min/+

mice was influenced by feeding diets

with varying carbohydrate sources and by anthocyanins.
Consumption of glucose led to the greatest small intestinal
adenoma number and burden in female mice, and the
stimulatory effect of glucose was inhibited by the addition
of dietary anthocyanins. The hypothesized mechanism of
action of anthocyanins is by inhibiting glucose absorption
and by acting as anti-inflammatory and anti-oxidative
agents.
Consumption of sucrose led to the greatest colonic
tumor size in both male and female mice. The stimulatory
effect of sucrose in this model is consistent with research
published previously by this laboratory. Additionally,
anthocyanins reduced average adenoma size in mice fed
sucrose. The hypothesized mechanism of action of
anthocyanins in the colon is their ability to act as antiinflammatory and antioxidative agents, and to reduce
inflammation-induced cell proliferation associated with
sucrose consumption.
In this study, 1.5 grams of approximately 50 percent
pure anthocyanins were added to each kilogram of diet. This
83

in effect resulted in an actual anthocyanin dose of 750 PPM
Assuming mice consumed 15 g per 100 g of bodyweight and had
a mean body weight of 21 grams from the start of dietary
treatment to termination, Apc

Min/+

mice would have consumed

an average of 2.4 mg/day of anthocyanins per day. This
would be the equivalent of 6.0 g/day of the cherry variety
used to extract the anthocyanins in this study, based on an
anthocyanin content of 0.4 mg/g (Kang et al., 2003).
However, it is important to note that a dose-dependence was
not investigated in this study, and doses lower than 750
ppm may be equally effective in this model.
In conclusion, these results support the health
promoting properties of anthocyanins; however, additional
research is needed to determine the mechanisms hypothesized
by these research findings and to investigate the potential
of anthocyanins to influence human cancer biomarkers.

84

REFERENCES

85

REFERENCES

Aggarwal BB, Shishodia S, Sandur SK, Pandey MK, Sethi G:
Inflammation and cancer: how hot is the link? Biochemical
Pharmacology 2006, 72:1605–21.
American Cancer Society: Colorectal Cancer Facts & Figures.
Atlanta; 2013:2006–2008.
American Cancer Society: American Cancer Society Guidelines
on Nutrition and Physical Activity for Cancer Prevention
Reducing the Risk of Cancer With Healthy Food Choices and
Physical Activity. 2012.
Barrett JC: Mechanisms of Multistep Carcinogenesis and
Carcinogen Risk Assessment. Environmental Health
Perspectives 1993, 100:9–20.
Beckwith AG, Zhang Y, Seeram NP, Cameron AC, Nair MG:
Relationship of Light Quantity and Anthocyanin Production
in Pennisetum setaceum Cvs. Rubrum and Red Riding Hood.
Journal of Agriculture and Food Chemistry 2004, 52:456-461.
Blando F, Gerardi C, Nicoletti I: Sour Cherry (Prunus
cerasus L) Anthocyanins as Ingredients for Functional
Foods. Journal of Biomedicine and Biotechnology 2004,
5:253–258.
Bobe G, Wang B, Seeram NP, Nair MG, Bourquin LD: Dietary
anthocyanin-rich tart cherry extract inhibits intestinal
tumorigenesis in APC(Min) mice fed suboptimal levels of
sulindac. Journal of Agricultural and Food Chemistry 2006,
54:9322–8.
Bostick RM, Potter JD, Kushi LH, Sellers TA, Steinmetz KA,
McKenzie DR, Gapstur SM, Folsom AR: Sugar, meat, and fat
intake, and non-dietary risk factors for colon cancer
incidence in Iowa women (United States). Cancer Causes and
Control 1994, 5:38–52.
Bristol J, Emmett P: Sugar, fat, and the risk of colorectal
cancer. British Medical Journal 1985, 291(NOVEMBER):1467–
1470.

86

Burt RW: Colon Cancer Screening. Gastroenterology 2000,
119:837–853.
Busserolles J, Rock E, Gueux E, Mazur A, Grolier P,
Rayssiguier Y: Short-term consumption of a high-sucrose
diet has a pro-oxidant effect in rats. British Journal of
Nutrition 2007, 87:337.
Caderni G,
M, Biggeri
dysplastic
crypt foci

Lancionia L, Luceria C, Gianninib A, Lodovicia
A, Dolaraa P: Dietary sucrose and starch affect
characteristics in carcinogen-induced aberrant
in rat colon. Cancer Letters 1997, 114:39–41.

Caderni G, Bianchini F, Mancina A, Spagnesi MT, Dolara P:
Effect of Dietary Carbohydrates on the Growth of Dysplastic
Crypt Foci in the Colon of Rats Treated with Effect of
Dietary Carbohydrates on the Growth of Dysplastic Crypt
Foci in the Colon of Rats Treated with 1, 2Dimethylhydrazine. Cancer Research 1991:3721–3725.
Caderni G., Dolara P., Spagnesi T., Lucerni C., Bianchini
F., Masterrandrea V. MG: Rats fed high starch diets have
lower colonic proliferation and faecal bile acids than high
sucrose-fed controls. Journal of Nutrition 1993, 123:704–
712.
Calabrese P, Mecklin J-P, Järvinen HJ, Aaltonen LA, Tavaré
S, Shibata D: Numbers of mutations to different types of
colorectal cancer. BMC Cancer 2005, 5:126.
Cao L: Influence of Apple Pomace and its Fractions on
Intestinal Tumor Development in APC Min Mice. Michigan
State University; 2012.
Centers for Disease Control and Prevention” Cancer
Prevention and Control. [http://www.cdc.gov/cancer/]. 2013.
Christofk HR, Vander Heiden MG, Harris MH, Ramanathan A,
Gerszten RE, Wei R, Fleming MD, Schreiber SL, Cantley LC:
The M2 splice isoform of pyruvate kinase is important for
cancer metabolism and tumour growth. Nature 2008, 452:230–
3.
Colotta F, Allavena P, Sica A, Garlanda C, Mantovani A:
Cancer-related inflammation, the seventh hallmark of
cancer: links to genetic instability. Carcinogenesis 2009,
30:1073–81.
87

Cooke D, Schwarz M, Boocock D, Winterhalter P, Steward WP,
Gescher AJ, Marczylo TH: Effect of cyanidin-3-glucoside and
an anthocyanin mixture from bilberry on adenoma development
in the ApcMin mouse model of intestinal carcinogenesis-relationship with tissue anthocyanin levels. International
Journal of Cancer 2006, 119:2213–20.
Corpet D, Stamp D, Medline A, Minkin S: Promotion of
colonic microadenoma growth in mice and rats fed cooked
sugar or cooked casein and fat. Cancer Research 1990:6955–
6958.
Coussens LM, Werb Z: Inflammation and cancer. Nature 2002,
420:860–7.
Crofford L: COX-1 and COX-2 tissue expression: implications
and predictions. The Journal of Rheumatology Supplement
1997, 49:15–19.
Douard V, Ferraris RP: Regulation of the fructose
transporter GLUT5 in health and disease. American Journal
of Physiology Endocrinology and Metabolism 2008, 295:E227–
37.
Erdman SE, Sohn JJ, Rao VP, Nambiar PR, Ge Z, Fox JG,
Schauer DB: CD4+ CD25+ Regulatory Lymphocytes Induce
Regression of Intestinal Tumors in Apc Min/+ Mice. American
Association for Cancer Research 2005, 65:3998–4004.
Faria A, Pestana D, Azevedo J, Martel F, de Freitas V,
Azevedo I, Mateus N, Calhau C: Absorption of anthocyanins
through intestinal epithelial cells - Putative involvement
of GLUT2. Molecular Nutrition & Food Research 2009,
53:1430–7.
Fearnhead NS, Britton MP, Bodmer WF, Hospital JR, Ox O: The
ABC of APC. Human Molecular Genetics 2001, 10:721–733.
Flood A, Rastogi T, Wirfält E, Mitrou PN, Reedy J, Subar
AF, Kipnis V, Mouw T, Hollenbeck AR, Leitzmann M, Schatzkin
A: Dietary patterns as identified by factor analysis and
colorectal cancer among middle-aged Americans. The American
Journal of Clinical Nutrition 2008, 88:176–84.

88

Franceschi S, Dal Maso L, Augustin L, Negri E, Parpinel M,
Boyle P, Jenkins DJ LVC: Dietary glycemic load and
colorectal cancer risk. Annals of Oncology 2001, 12:173–
178.
Fung T, Hu FB, Fuchs C, Giovannucci E, Hunter DJ, Stampfer
MJ, Colditz GA, Willett WC: Major dietary patterns and the
risk of colorectal cancer in women. Archives of Internal
Medicine 2003, 163:309–14.
Greenhough A, Smartt HJM, Moore AE, Roberts HR, Williams
AC, Paraskeva C, Kaidi A: The COX-2/PGE2 pathway: key roles
in the hallmarks of cancer and adaptation to the tumour
microenvironment. Carcinogenesis 2009, 30:377–86.
Grivennikov SI, Greten FR, Karin M: Immunity, inflammation,
and cancer. Cell 2010, 140:883–99.
Gryfe R, Swallow C, Bapat B, Redston M, Gallinger S,
Couture J: Current Problems in Cancer: Molecular Biology of
Colorectal Cancer. Current Problems in Cancer 1997, 21.
Hagiwara A, Miyashita K, Nakanishi T, Sano M, Tamano S,
Kadota T, Koda T, Nakamura M, Imaida K, Ito N, Shirai T:
Pronounced inhibition by a natural anthocyanin , purple
corn color , associated colorectal carcinogenesis in male
F344 rats pretreated with 1,2-dimethylhydrazine. Cancer
Letters 2001, 171.
Hanahan D, Weinberg RA: Hallmarks of cancer: the next
generation. Cell 2011, 144:646–674.
Harborne JB: Spectral methods of characterizing
anthocyanins. Biochem Journal 1958, 70(1): 22-28.
Hardt PD, Mazurek S, Toepler M, Schlierbach P, Bretzel RG,
Eigenbrodt E, Kloer HU: Faecal tumour M2 pyruvate kinase: a
new, sensitive screening tool for colorectal cancer.
British Journal of Cancer 2004, 91:980–4.
Hawkey CJ: New drug classes COX-2 inhibitors. The Lancet
1999, 353:307–314.
Ichiyanagi T, Shida
Bioavailability and
bilberry (Vaccinium
of Agricultural and

Y, Rahman MM, Hatano Y, Konishi T:
tissue distribution of anthocyanins in
myrtillus L.) extract in rats. Journal
Food Chemistry 2006, 54:6578–87.
89

Issa AY, Volate SR, Muga SJ, Nitcheva D, Smith T, Wargovich
MJ: Green tea selectively targets initial stages of
intestinal carcinogenesis in the AOM-ApcMin mouse model.
Carcinogenesis 2007, 28:1978–84.
Jackson Laboratory. Body weight information.
[http://jaxmice.jax.org/support/weight/000664.html]. 2013.
Jasperson KW, Tuohy TM, Neklason DW, Burt RW: Hereditary
and familial colon cancer. Gastroenterology 2010, 138:2044–
58.
Jing P, Bomser J a, Schwartz SJ, He J, Magnuson B a, Giusti
MM: Structure-function relationships of anthocyanins from
various anthocyanin-rich extracts on the inhibition of
colon cancer cell growth. Journal of Agricultural and Food
Chemistry 2008, 56:9391–8.
Johnston K, Sharp P, Clifford M, Morgan L: Dietary
polyphenols decrease glucose uptake by human intestinal
Caco-2 cells. FEBS letters 2005, 579:1653–7.
Kang S-Y, Seeram NP, Nair MG, Bourquin LD: Tart cherry
anthocyanins inhibit tumor development in ApcMin mice and
reduce proliferation of human colon cancer cells. Cancer
Letters 2003, 194:13–19.
Karin M: Nuclear factor-kappaB in cancer development and
progression. Nature 2006, 441:431–6.
Karin M, Greten FR: NF-kappaB: linking inflammation and
immunity to cancer development and progression. Nature
Reviews Immunology 2005, 5:749–59.
Kinzler KW, Vogelstein B: Lessons from hereditary
colorectal cancer. Cell 1996, 87:159–70.
Kirakosyan A, Seymour EM, Noon K, Kaufman P, Warber S,
Bolling S: Bioavailability of Tart Cherry Antioxidants in
Human Subjects. Free Radical Biology and Medicine 2010,
49:S221.
Kobayashi Y, Suzuki M, Satsu H, Arai S, Hara Y, Suzuki K,
Miyamoto Y, Shimizu M: Green tea polyphenols inhibit the
sodium-dependent glucose transporter of intestinal
epithelial cells by a competitive mechanism. Journal of
Agricultural and Food Chemistry 2000, 48:5618–23.
90

Koushik A, Hunter DJ, Spiegelman D, Beeson WL, van den
Brandt P a, Buring JE, Calle EE, Cho E, Fraser GE,
Freudenheim JL, Fuchs CS, Giovannucci EL, Goldbohm RA,
Harnack L, Jacobs DR, Kato I, Krogh V, Larsson SC,
Leitzmann MF, Marshall JR, McCullough ML, Miller AB,
Pietinen P, Rohan TE, Schatzkin A, Sieri S, Virtanen MJ,
Wolk A, Zeleniuch-Jacquotte A, Zhang SM, et al.: Fruits,
vegetables, and colon cancer risk in a pooled analysis of
14 cohort studies. Journal of the National Cancer Institute
2007, 99:1471–83.
Kristiansen E, Thorpe IM: Influence of different diets on
development of DMH‐induced aberrant crypt foci and colon
tumor incidence in Wistar rats. Nutr Cancer 1995, 23:151–
159.
Kristiansen E, Meyer O, Thorup I: Refined carbohydrate
enhancement of aberrant crypt foci (ACF) in rat colon
induced by the food-borne carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). Cancer Letters 1996, 105:147–
151.
Kumar Y, Tapuria N, Kirmani N, Davidson BR: Tumour M2pyruvate kinase: a gastrointestinal cancer marker. European
Journal of Gastroenterology & Hepatology 2007, 19:265–76.
Kwong LN, Dove WF: APC and its modifiers in colon cancer.
Advances in Experimental Medicine and Biology 2009, 656:85–
106.
La Vecchia C, Negri E, Decarli A, Avanzo BD: A Case-control
Study of Diet and Colo-rectal Cancer in Northern Italy.
International Journal of Cancer 1988, 498:492–498.
Markowitz SD, Bertagnolli MM: Molecular Basis of Colorectal
Cancer. The New England Journal of Medicine 2009:2449–2460.
Matuschek MC, Hendriks WH, McGhie TK, Reynolds GW: The
jejunum is the main site of absorption for anthocyanins in
mice. The Journal of Nutritional Biochemistry 2006, 17:31–
6.
McConnell BB, Yang VW: The Role of Inflammation in the
Pathogenesis of Colorectal Cancer. Molecular Biology 2009,
2.

91

McGhie TK, Walton MC: The bioavailability and absorption of
anthocyanins: towards a better understanding. Molecular
Nutrition & Food Research 2007, 51:702–13.
Mehlen P, Fearon ER: Role of the dependence receptor DCC in
colorectal cancer pathogenesis. Journal of Clinical
Oncology: Official Journal of the American Society of
Clinical Oncology 2004, 22:3420–8.
Michaud DS, Fuchs CS, Liu S, Willett WC, Colditz GA,
Giovannucci E: Dietary glycemic load, carbohydrate, sugar,
and colorectal cancer risk in men and women. Cancer
Epidemiology, Biomarkers & Prevention: a publication of the
American Association for Cancer Research, cosponsored by
the American Society of Preventive Oncology 2005, 14:138–
47.
Miller PE, Lesko SM, Muscat JE, Lazarus P, Hartman TJ:
Dietary patterns and colorectal adenoma and cancer risk: a
review of the epidemiological evidence. Nutrition and
Cancer 2010, 62:413–24.
Miyazawa T, Nakagawa K, Kudo M, Muraishi K, Someya K:
Direct intestinal absorption of red fruit anthocyanins,
cyanidin-3-glucoside and cyanidin-3,5-diglucoside, into
rats and humans. Journal of Agricultural and Food Chemistry
1999, 47:1083–91.
Mohammed A, Janakiram NB, Li Q, Choi C-I, Zhang Y, Steele
VE, Rao CV: Chemoprevention of colon and small intestinal
tumorigenesis in APC(Min/+) mice by licofelone, a novel
dual 5-LOX/COX inhibitor: potential implications for human
colon cancer prevention. Cancer Prevention Research 2011,
4:2015–26.
Mollersen L, Paulsen JE, Alexander J: Loss of
Heterozygosity and Nonsense Mutations in Apc in
Azoxymenthane-induced Colonic Tumours in Min Mice.
Anticancer Research, 2004 24:2595-2600.
Moorghen M, Ince P, Finney KJ, Sunter JP, Appleton DR,
Watson AJ: A protective effect of sulindac against
chemically-induced primary colonic tumours in mice. The
Journal of Pathology 1988, 156:341–7.

92

Mulabagal V, Lang GA, DeWitt DL, Dalavoy SS, Nair MG:
Anthocyanin content, lipid peroxidation and cyclooxygenase
enzyme inhibitory activities of sweet and sour cherries.
Journal of Agricultural and Food Chemistry 2009, 57:1239–
46.
Murkovic M, Mulleder U, Adam U, Pfannhauser W: Detection of
anthocyanins from elderberry juice in human urine. Journal
of the Science of Food and Agriculture 2001, 81:934–937.
National Cancer Institute. Cancer Statistics
[http://www.cancer.gov/statistics]. 2013.
Nayak SP, Sasi MP, Sreejayan MP, Mandal S: A Case-control
Study of Roles of Diet in Colorectal Carcinoma in a South
Indian Population. Asian Pacific Journal of Cancer
Prevention 2009, 10:565–568.
Ohnishi R, Ito H, Kasajima N, Kaneda M, Kariyama R, Kumon
H, Hatano T, Yoshida T: Urinary Excretion of Anthocyanins
in Humans after Cranberry Juice Ingestion. Bioscience,
Biotechnology, and Biochemistry 2006, 70:1681–1687.
Passamonti S, Vrhovsek U, Mattivi F: The interaction of
anthocyanins with bilitranslocase. Biochemical and
Biophysical Research Communications 2002, 296:631–6.
Passamonti S, Vrhovsek U, Vanzo A, Mattivi F: The stomach
as a site for anthocyanins absorption from food. FEBS
Letters 2003, 544:210–213.
Pietta PG: Flavonoids as antioxidants. Journal of Natural
Products 2000, 63:1035–42.
Pitot HC, Goldsworthy T, Moran S: The natural history of
carcinogenesis: implications of experimental carcinogenesis
in the genesis of human cancer. Journal of Supramolecular
Structure and Cellular Biochemistry 1981, 17:133–46.
Pollard M, Luckert PH: Prevention and Treatment of Primary
Intestinal Tumors in Rats by Piroxicam Prevention and
Treatment of Primary Intestinal Tumors in Rats by
Piroxicam. Cancer Research 1989:6471–6473.

93

Pollock CB, Shirasawa S, Sasazuki T, Kolch W, Dhillon AS:
Oncogenic K-RAS is required to maintain changes in
cytoskeletal organization, adhesion, and motility in colon
cancer cells. Cancer Research 2005, 65:1244–50.
Potter JD, Slattery ML, Bostick RM, Gapstur SM: Colon
cancer: a review of the epidemiology. Epidemiologic Reviews
1993, 15:499–545.
Poulsen M, Mølck a M, Thorup I, Breinholt V, Meyer O: The
influence of simple sugars and starch given during pre- or
post-initiation on aberrant crypt foci in rat colon. Cancer
Letters 2001, 167:135–43.
Powell KE: Effects of sucrose, glucose, and fructose
consumption. Michigan State University; 2011.
Prior RL: Fruits and vegetables in the prevention of
cellular oxidative damage. The American Journal of Clinical
Nutrition 2003, 78:570–578.
Quina FH, Moreira PF, Vautier-Giongo C, Rettori D,
Rodrigues RF, Freitas AA, Silva PF, Maçanita AL:
Photochemistry of anthocyanins and their biological role in
plant tissues. Pure and Applied Chemistry 2009, 81:1687–
1694.
Reddy BS, Rao CV, Rivenson A, Kelloff G: Inhibitory effect
of aspirin on azoxymethane-induced colon carcinogenesis in
F344 rats. Carcinogenesis 1993, 14:1493–7.
Reddy BS, Maruyama H, Kelloff G, Muruvanni H, Kelloft G:
Dose-related Inhibition of Colon Carcinogenesis by Dietary
Piroxicam, a Nonsteroidal Anti-inflammatory Drug, during
Different Stages of Rat Colon Tumor Development. Cancer
Research 1987:5340–5346.
Reeves PG, Nielsen FH, Fahey GC: AIN-93 purified diets for
laboratory rodents: final report of the American Institute
of Nutrition ad hoc writing committee on the reformulation
of the AIN-76A rodent diet. The Journal of Nutrition 1993,
123:1939–51.
Riboli E, Norat T: Epidemiologic evidence of the protective
effect of fruit and vegetables on cancer risk. The American
Journal of Clinical Nutrition 2003, 78.

94

Sandur SK, Deorukhkar A, Pandey MK, Pabón AM, Shentu S,
Guha S, Aggarwal BB, Krishnan S: Curcumin modulates the
radiosensitivity of colorectal cancer cells by suppressing
constitutive and inducible NF-kappaB activity.
International Journal of Radiation Oncology, Biology,
Physics 2009, 75:534–42.
Sano H, Kawahito Y, Wilder RL, Hashiramoto A, Mukai S, Asai
K, Kimura S, Kato H, Kondo M, Hla T: Expression of
Cyclooxygenase-1 and -2 in Human Colorectal Cancer. Cancer
Research 1995, 55:3785–3789.
Schulze MB, Hoffmann K, Manson JE, Willett WC, Meigs JB,
Weikert C, Heidemann C, Colditz G a, Hu FB: Dietary
pattern, inflammation, and incidence of type 2 diabetes in
women. The American Journal of Clinical Nutrition 2005,
82:675–84; quiz 714–5.
Seeram NP, Momin R a, Nair MG, Bourquin LD: Cyclooxygenase
inhibitory and antioxidant cyanidin glycosides in cherries
and berries. Phytomedicine: International Journal of
Phytotherapy and Phytopharmacology 2001, 8:362–9.
Seeram NP, Zhang Y, Nair MG: Inhibition of proliferation of
human cancer cells and cyclooxygenase enzymes by
anthocyanidins and catechins. Nutrition and Cancer 2003,
46:101–6.
Sethi G, Sung B, Aggarwal BB: Nuclear factor-kappaB
activation: from bench to bedside. Experimental Biology and
Medicine 2008, 233:21–31.
Seymour EM, Lewis SK, Urcuyo-llanes DE, Tanone II,
Kirakosyan A, Kaufman PB, Bolling SF, Al SET: Regular Tart
Cherry Intake Alters Abdominal Adiposity, Adipose Gene
Transcription, and Inflammation in Obesity-Prone Rats Fed a
High Fat Diet. Journal of Medicinal Food 2009, 12:935–942.
Skinner SA, Penney AG OP: Sulindac inhibits the rate of
growth and appearance of colon tumors in the rat. Arch Surg
1991, 126:1094–6.

95

Slattery ML, Benson J, Berry TD, Slattery L, Edwards L,
Potter JD, Dennis T, Caan BJ: Dietary sugar and colon
cancer. Cancer Epidemiology, Biomarkers & Prevention: a
publication of the American Association for Cancer
Research, cosponsored by the American Society of Preventive
Oncology 1997, 6:677–684.
Slattery ML, Boucher KM, Caan BJ, Potter JD, and Ma K-H:
Eating Patters and Risk of Colon Cancer. American Journal
of Epidemiology 1997, 148(1):4-16.
Sørensen LB, Raben A, Stender S, Astrup A: Effect of
sucrose on inflammatory markers in overweight humans. The
American Journal of Clinical Nutrition of Clinical
Nutrition 2005, 82:421–7.
Stamp D, Zhang XM, Medline A, Bruce WR, Archer MC: Sucrose
enhancement of the early steps of colon carcinogenesis in
mice. Carcinogenesis 1993, 14:777–9.
Su, Li-Kuo, Kinzler, Kenneth W., Vogelstein, Bert,
Preisinger, Antonette C., Moser, Amy R., Luongo Cindy,
Gould, Karen A., Dove WF: Multiple Intestinal Neoplasia
Caused by a Mutation in the Murine Homolog of the APC Gene.
Science 1992, 256:668–670.
Suzui M, Okuno M, Tanaka T, Nakagama H, Moriwaki H:
Enhanced colon carcinogenesis induced by azoxymethane in
min mice occurs via a mechanism independent of beta-catenin
mutation. Cancer Letters 2002, 183:31–41.
Taketo MM: COX-2 and colon cancer. Inflammation Research
1998, 47 Suppl 2:S112–6.
Talavéra S, Felgines C, Texier O, Besson C, Lamaison J-L,
Rémésy C: Anthocyanins are efficiently absorbed from the
stomach in anesthetized rats. The Journal of Nutrition
2003, 133:4178–82.
Tall JM, Seeram NP, Zhao C, Nair MG, Meyer RA, Raja SN:
Tart cherry anthocyanins suppress inflammation-induced pain
behavior in rat. Behavioural Brain Research 2004, 153:181–
8.
Tammali R, Ramana K V, Srivastava SK: Aldose reductase
regulates TNF-a-induced PGE2 production in human colon
cancer cells. Cancer Letters 2007, 252:299–306.
96

Taubes G: Unraveling the Obesity-Cancer Connection. Science
2012, 335:28–32.
Terry P, Hu FB, Hansen H, Wolk A: Prospective study of
major dietary patterns and colorectal cancer risk in women.
American Journal of Epidemiology 2001, 154:1143–9.
Terry P, Giovannucci E, Michels KB, Bergkvist L, Hansen H,
Holmberg L, Wolk A: Fruit , Vegetables , Dietary Fiber, and
Risk of Colorectal Cancer. Journal of the National Cancer
Institute 1998:525–533.
Terzić J, Grivennikov S, Karin E, Karin M: Inflammation and
colon cancer. Gastroenterology 2010, 138:2101–2114.e5.
Thun MJ, Namboodiri MM, Calle EE, Flanders WD, Heath CW:
Aspirin Use and Risk of Fatal Cancer. Cancer Research
1993:1322–1327.
Thun, Michael J., Namboodiri, Mohan., Heath CW: Aspirin Use
and Reduced Risk of Fatal Colon Cancer. The New England
Journal of Medicine 1991, 325:1593–1596.
Tsuda T, Horio F, Osawa T: Absorption and metabolism of
cyanidin 3-O-beta-D-glucoside in rats. FEBS letters 1999,
449:179–82.
Vogelstein B, Fearon E: Genetic alterations during
colorectal-tumor development. The New England Journal of
Medicine 1988, 319:525–532.
Waddell WR, Ganser GF, Loughry RW: Sulindac for Polyposis
of the Colon. The American Journal of Surgery 1989,
157(January):175–179.
Wang B: Modulation of Intestinal Tumorigenesis by Dietary
Carbohydrate and Protein Sources in APC Min Mice. Michigan
State University; 2005.
Wang B, Bobe G, LaPres JJ, Bourquin LD: High sucrose diets
promote intestinal epithelial cell proliferation and
tumorigenesis in APC(Min) mice by increasing insulin and
IGF-I levels. Nutrition and Cancer 2009, 61:81–93.

97

Wang H, Nair MG, Strasburg GM, Chang YC, Booren a M, Gray
JI, DeWitt DL: Antioxidant and anti-inflammatory activities
of anthocyanins and their aglycon, cyanidin, from tart
cherries. Journal of Natural Products 1999, 62:294–6.
Wang H, Nair MG, Iezzoni AF, Strasburg GM, Booren AM, Gray
JI: Quantification and Characterization of Anthocyanins in
Balaton Tart Cherries. Journal of Agricultural and Food
Chemistry 1997, 45:2556–2560.
Wang L-S, Stoner GD: Anthocyanins and their role in cancer
prevention. Cancer Letters 2008, 269:281–90.
Wright EM: The Intestinal Na+/Glucose Cotransporter. Annual
Review of Physiology 1993, 55:575–589.
Yang K, Fan K, Kurihara N, Shinozaki H, Rigas B, Augenlicht
L, Kopelovich L, Edelmann W, Kucherlapati R, Lipkin M:
Regional response leading to tumorigenesis after sulindac
in small and large intestine of mice with Apc mutations.
Carcinogenesis 2003, 24:605–611.
Yang VW, Shields JM, Hamilton SR, Polyposis A, Spannhake
EW, Hubbard WC, Hylind LM, Robinson CR, Giardiello FM:
Size-dependent Increase in Prostanoid Levels in Adenomas of
Patients with Familial Adenomatous Polyposis Size-dependent
Increase in Prostanoid Levels in Adenomas of Patients with
Familial. Cancer Research 1998, 58:1750–1753.
Yi W, Fischer J, Krewer G, Akoh CC: Phenolic compounds from
blueberries can inhibit colon cancer cell proliferation and
induce apoptosis. Journal of Agricultural and Food
Chemistry 2005, 53:7320–9.

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