The effect of exercise on biological pathways in ApcMin/+ mouse intestinal polyps

1,3Kristen A. Baltgalvis, 2,3Franklin G. Berger, 2,3Maria M. Pena, 1J. Mark Davis, and 1,3James A. Carson

1Division of Applied Physiology, Exercise Science Department,2Department of Biological Sciences3Center for Colon Cancer Research,

University of South Carolina, Columbia SC

Running title: ApcMin/+ mice and exercise

Corresponding author:

James A. Carson, Ph.D.University of South CarolinaDepartment of Exercise ScienceRm 405A Public Health Research Building921 Assembly StreetColumbia SC 29208Office Phone: 803-777-0809Lab Phone: 803-777-0142Fax: 803-777-8422carsonj@sc.edu

Page 1 of 31 Articles in PresS. J Appl Physiol (January 31, 2008). doi:10.1152/japplphysiol.00955.2007

Copyright © 2008 by the American Physiological Society.

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ABSTRACT

Many epidemiological studies have demonstrated that level of exercise is associated with

reduced colorectal cancer risk. Treadmill training can decrease ApcMin/+ mouse intestinal polyp

number and size, but the mechanisms remain unclear. Understanding the molecular changes in

the tumor following exercise training may provide insight on the mechanism by which exercise

decreases ApcMin/+ mouse polyp formation and growth. The purpose of this study was to

determine if exercise can modulate ApcMin/+ mouse intestinal polyp cellular signaling related to

tumor formation and growth. Male ApcMin/+ mice were randomly assigned to Control (n=20) or

Exercise (n=20) treatment groups. Exercised mice ran on a treadmill at a moderate intensity (18

m/min, 60 min, 6 d/wk, 5% grade) for 9 weeks. Polyps from ApcMin/+ mice were used to quantify

markers of polyp inflammation, apoptosis, and β-catenin signaling. Exercise decreased the

number of macrophages in large polyps by 35%. Related to apoptosis, exercise decreased the

number of TUNEL positive cells by 73% in all polyps. Bax protein expression in polyps was

decreased 43% by exercise. Β-catenin phosphorylation was elevated 3.3-fold in polyps from

exercised mice. Moderate-intensity exercise training alters cellular pathways in ApcMin/+ mouse

polyps and these changes may be related to the exercise-induced reduction in polyp formation

and growth.

KEYWORDS: COLON CANCER, PHYSICAL ACTIVITY, INFLAMMATION, APOPTOSIS,

β-CATENIN

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INTRODUCTION

There is a strong inverse relationship between exercise and colon cancer risk in both men

and women (23). Rodent models of colorectal cancer have been used to confirm that exercise

can prevent colorectal tumor formation and progression (1, 9, 28, 40, 47). ApcMin/+ mice are a

widely accepted model used to study interventions that prevent colorectal cancer. These mice

have a nonsense mutation at codon 850 in the Apc (Adenomatous Polyposis Coli) gene which

predisposes them to both small and large intestinal adenomas (31). Regular moderate-intensity

exercise can reduce the ApcMin/+ mouse intestinal polyp burden (9, 28). Many theorized

mechanisms for the protective effect of exercise on colorectal cancer risk include a decreased

gastrointestinal transit time, an altered inflammatory state, and a reduction in circulating growth

factors (39). However, the biological mechanisms related to exercise-induced colon cancer

prevention remain poorly understood. Systemic changes in exercised ApcMin/+ mice, such as

insulin-like growth factor-1 (IGF-1), leptin, corticosterone, and inflammatory cytokines, have

been investigated and correlated with changes in the ApcMin/+ mouse polyp burden (7-9, 28).

However, direct exercise effects on biological changes within intestinal polyps have not been

investigated in the ApcMin/+ mouse.

Tumors arise from many different tissue types, but all tumors have similar characteristics.

All tumors produce growth factors, escape apoptosis, and have uncontrolled cell division (12).

The growth of tumors can also be attributed to chronic inflammation in both human and animal

models of colon cancer (18). Inflammatory cells, such as macrophages, are recruited to the

tumor assist in tumor growth, proliferation, and metastasis (10, 25). Tumor-associated

macrophages provide tumors with survival factors, such as interleukin-1β, interleukin-6 (22), and

cyclooxygenase-2 (COX-2) (16). COX-2 is an inducible enzyme that is responsible for

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prostaglandin synthesis and its activity is elevated in colon tumors (44). Inhibition of COX-2

can prevent or cause regression of colorectal polyps in Familial Adenomatous Polyposis (FAP)

patients and in ApcMin/+ mice (34, 44, 46).

The main consequence of an Apc mutation is the constitutive activation of nuclear

localization of β-catenin and activation of genes associated with cell cycle progression (4, 6).

Interestingly, intestinal Apc deletion promotes apoptosis and ApcMin/+ mice demonstrate an

increase in apoptosis-related proteins compared to wild-type intestinal epithelia (35, 43). The

Wnt pathway also interacts with the phosphatidylinositol 3-kinase/Akt pathway, which is

activated in a number of tumor types, including human colon cancer (11, 37, 48) and in ApcMin/+

mouse intestinal polyps (20, 30, 35). Growth factors, such as insulin or IGFs, activate Akt. Akt

can regulate β-catenin nuclear localization, leading to polyp formation (26, 48). Exercise may

prevent ApcMin/+ mouse polyp formation by inhibition of the Apc→β-catenin signaling pathway.

Exercise has been shown to decrease both the number and size of polyps in ApcMin/+ mice

(9, 28). While there has been considerable focus on the ability of exercise to prevent polyp

formation, the ability of exercise to block or repress the growth of existing polyps has important

health implications and has not been well described in ApcMin/+ mice. The loss of Apc leads to

nuclear β-catenin localization, which is responsible for adenoma formation. Local growth

factors signal through Akt can also activate this pathway by phosphorylating and inactivating

GSK-3β, which also leads to β-catenin activation. Once adenomas are formed, immune cells are

often recruited to tumors to fight the immortalized cells. However, tumor-associated

macrophages are associated with tumor growth and the secretion of inflammatory cytokines (25).

Apoptosis, or programmed cell death, is typically evaded by tumors and leads to uncontrolled

tumor growth (12). Exercise can regulate inflammation (49, 51), apoptosis (45), β-catenin (2, 42,

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50), and Akt signaling (2, 5, 19, 42) in other tissues. It is not known whether exercise can

regulate these critical pathways in ApcMin/+ mouse intestinal polyps. The primary purpose of the

current study was to determine if exercise alters signaling in polyps related to inflammation and

growth. We hypothesized that exercise would attenuate macrophage infiltration and COX-2

expression in intestinal polyps from ApcMin/+ mice, when compared to sedentary mice.

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METHODS

Animals. ApcMin/+ male mice were supplied by the Colorectal Cancer Research Center Breeding

Core Facility at the University of South Carolina as previously described (28). A subset of

animals (Control; n=13 and Exercise; n=12) used in this study for immunohistochemical analysis

have previously had their polyp counts published (28). All data presented in the current paper

has not been published previously. Due to polyp counts being performed on formalin fixed

tissue methodological limitations required a second group on mice to isolate polyps for protein

expression analysis (see methodology). This additional set of control (n=7) and exercised (n=8)

mice underwent an identical exercise stimulus as the first set of mice, and the exercise treatments

were performed consecutively with animals from the same breeding colony at the University of

South Carolina as those used in the initial study examining polyp counts. All animal

experimentation was approved by the University of South Carolina’s Institutional Animal Care

and Use Committee.

Treadmill protocol. Male ApcMin/+ mice (Control (n=20) and Exercise (n=20)) were exercised as

previously described (28). This moderate-intensity exercise protocol has previously shown to

reduce male ApcMin/+ mouse polyp number. Briefly, 3.5-wk-old male mice ran on the treadmill

(18 m/min; 60 min/d; 6 d/wk; 5% grade) for a total of 9 weeks. All training took place at the

beginning of the dark cycle with the guidance of a red light. Mice were motivated to run by

gentle hand prodding. Controls were kept next to the treadmill, but remained in their cages

during training. All mice were sacrificed at 13 weeks of age, at least 36 hours after the last bout

of exercise. This exercise training protocol induces a significant increase in gastrocnemius

muscle citrate synthase activity in ApcMin/+ mice (28).

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Tissue collection. Mice were given a subcutaneous injection of ketamine/xylazine/acepromazine

cocktail (1.4 ml/kg BW). The small intestines were carefully dissected distally to the stomach

and proximal to the cecum. The large intestine was removed from the distal end of the cecum to

the anus. Mesentery tissue was removed with tweezers and the small intestine was cut into four

equal sections. All intestinal sections were flushed with PBS, opened longitudinally, and

flattened with a cotton swab. The distal end of the ileum was fixed in 10% buffered formalin for

24 hours and transferred to 70% ethanol for histochemical analysis on all animals (n=12-13 per

group). With an additional set of animals (n=7-8 per group), animals were anesthetized and the

intestinal tract was dissected similarly, except ileum intestinal polyps were dissected from the

intestinal tract and frozen in liquid nitrogen for protein analysis.

Western Blotting. Briefly, frozen polyps (n=7-8 animals per treatment group) were homogenized

in Mueller buffer and protein concentration was determined by the Bradford method, as

previously described (27). Crude polyp homogenates (10-20 µg) were fractionated on 8%-12%

polyacrylamide gels. Gels were transferred to PVDF membranes overnight and Ponceau staining

was used to visually confirm the gel transfer and equal loading. Membranes were blocked in 5%

TBST milk for 1 h at room temperature. Primary antibodies for phosphorylated β-catenin (Ser

33/37 Thr 41), total β-catenin, phosphorylated Akt (Ser 373), total Akt (Cell Signaling), and Bax

(Calbiochem), were incubated at dilutions of 1:500 to 1:1000 overnight at 4 °C in 1% TBST

milk. Secondary anti-rabbit or anti-mouse IgG conjugated secondary antibodies (Amersham

Biosciences) were incubated with the membranes at 1:2000 to 1:5000 dilutions for 1 hour in 1%

TBST milk. Enhanced chemiluminescence (Amersham Biosciences) was used to visualize the

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antibody-antigen interactions and developed by autoradiography (Kodak, Biomax, MR).

Digitally scanned blots were analyzed by measuring the integrated optical density (IOD) of each

band using digital imaging software (Scion Image, Frederick, MD).

Immunohistochemistry. Formalin-fixed, paraffin-embedded intestinal sections were Swiss-rolled

and were cut on a microtome in 4 µm sections (n=12-13 animals per treatment group). Sections

were deparaffinized in xylene and rinsed in 100% ethanol. Peroxidase activity was squelched

with 6% H2O2 in methanol for 30 minutes. Antigen retrieval was accomplished with 0.1%

bovine trypsin for 30 min at 37 ºC. Sections were blocked for 30 minutes in rabbit serum. Slides

were incubated 1:100 with β-catenin (BD Transduction Laboratories), F4/80 (Serotec), or COX-

2 (Caymen Chemical) for 1 h at 37 ºC. Slides were rinsed in PBS and incubated 1:200 with anti-

rabbit peroxidase-conjugated antibody for 1 h 37 ºC. Color detection was visualized with an

Vectastain ABC (Avidin-Biotinylated Enzyme Complex) detection kit and DAB (3,3’-

diaminobenzidine) (Vector laboratories, Burlingame, CA). β-catenin-accumulated crypts within

polyps or COX-2 or F4/80-positive cells were counted for each polyp on a microscope at a 400x

magnification by a technician blinded to the treatments. Polyps with <5 β-catenin-accumulated

crypts was considered a small polyp while polyps >5 β-catenin-accumulated crypts was

considered large. The number of these small and large polyps was average for each animal. For

COX-2 and F4/80 analyses, cells were counted for all polyps within an animal and averaged per

animal. Data were also examined by polyp size ( <1 mm or > 1 mm) when appropriate.

TUNEL assay. Apoptotic cells were detected using a kit purchased from Chemicon

International. Swiss-rolled, formalin-fixed, paraffin-embedded intestinal sections (4 µm) were

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stained for apoptotic cells according to manufacturer’s instructions. TUNEL positive cells were

counted on a microscope at a 400x magnification by a technician blinded to the treatments. Cells

were counted for all polyps within an animal and averaged per animal, as well as stratified by

polyp size.

Statistical analysis. Results are reported as means ± standard errors. All variables were

analyzed with independent t-tests. Histological analyses of polyps were analyzed with

independent t-tests between Control and Exercise mice for all polyps and also within each polyp

size when appropriate. T-tests were also performed within the cohorts of Control mice and

Exercise mice examining body mass and spleen mass to ensure homogeneity between the 2

experiments. The level of statistical significance was accepted as p < 0.05.

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RESULTS

Animal characteristics. We have previously reported that this moderate-intensity treadmill

running protocol decreases intestinal polyp number 29% (28), and a subset of these animals were

used for immunohistological analyses (n=12-13 mice per group) in the current study. A second

set of control and exercised mice were also used in the current study in order to analyze protein

expression within polyps (Control; n=7 and Exercise; n=8). To ensure homogeneity amongst the

Control mice and Exercise mice between the 2 experiments, t-tests were performed on body

mass and spleen mass (an indirect marker of polyp burden). There were no significant

differences in body mass amongst the Control mice (24.3 ± 0.6 vs. 24.8 ± 0.6 g; p=0.143) or

Exercise mice (24.3 ± 0.5 vs. 23.1 ± 0.7g; p=0.159) between the 2 experimental groups. Spleen

weight also did not differ between Control (167 ± 16 vs. 132 ± 26 mg; p=0.230) and Exercise

mice (91 ± 9 vs. 73 ± 7 mg; p=0.169), suggesting genetic homogeneity amongst the groups. In

these additional mice, spleen weight did decrease with exercise training (132 ± 26 vs. 73 ± 7 mg;

p=0.03), similar to our previous study. While some exercise training protocols can induce

muscle hypertrophy and promote fat loss, this training protocol was not sufficient to alter body

weight (24.0 ± 0.5 vs. 22.5 ± 0.6 g; p=0.064), gastrocnemius muscle weight (113 ± 6 vs. 103 ± 6

mg; p=0.267), or epididymal fat pad mass (214 ± 24 vs. 251 ± 19 mg; p=0.234) between Control

and Exercise ApcMin/+ mice.

Polyp inflammation. The effect of exercise on tumor-associated macrophage infiltration was

identified by F4/80 immunohistochemistry in the distal ileum of ApcMin/+ mice (Figures 1A &

1B). Nine weeks of treadmill training produced a 35% decrease in F4/80-positive cells in polyps

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(Figure 1C; p=0.0.010). When polyps were stratified by size, only large polyps >1mm in

diameter showed fewer F4/80 cells in exercised polyps (20 ± 2 vs. 13 ± 3; p=0.037).

To determine if exercise alters polyp COX-2 expression, immunohistochemistry was also

used to identify COX-2-positive cells within polyps located in the distal ileum (Figures 2A &

2B). Intestinal polyps were categorized as >1 mm or <1 mm in diameter. There were no

differences in the number of COX-2-positive cells within polyps (p=0.985). Total COX-2

protein was also measured in polyps by western blot (Figure 5A). Total COX-2 protein did not

change in polyps with exercise training (Figure 5B; p=0.835).

Apoptosis. TUNEL staining was used to localize apoptotic cells within polyps (Figures 3A &

3B). Exercise significantly reduced TUNEL positive cells 73% (Figure 3C; p=0.009). When

stratified by size, exercise reduced the incidence of TUNEL positive cells in small diameter

polyps (>1 mm) by 78% (p=0.001). Bax is a pro-apoptotic protein whose induction normally

coincides with increased apoptosis. The relative concentration of Bax protein was measured in

polyps by Western blot analysis (Figures 5A & 5B). Exercise significantly reduced polyp Bax

protein concentration 43% (p=0.003).

β-catenin. β-catenin expression immunohistochemistry was performed on Swiss-rolled sections

of the distal ileum in both Control and Exercise mice (Figures 4A & 4B). β-catenin-accumulated

crypts (BCACs) are precancerous lesions found in the ApcMin/+ mouse (15) and this similar

pattern of β-catenin expression is found in adenomas (21). β-catenin positive crypts within

polyps were counted for each polyp. Polyps were also classified by having a small number of β-

catenin foci (<5 foci) or a large number of foci (>5). Exercise had no effect on the number of β-

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catenin-accumulated crypts in polyps with a small number of foci (p=0.275) or a large number of

foci (p=0.420; Figure 4C).

Protein extracts from isolated polyps snipped from the small intestine were also examined

for total and phosphorylated β-catenin levels (Figure 5A). Exercise appears to affect β-catenin

phosphorylation, rather than total expression levels. Exercise had no affect on polyp β-catenin

protein concentration (p=0.142), but exercise induced phosphorylated β-catenin (Ser 33/37 Thr

41) 2.2-fold (p=0.004) and the ratio of phosphorylated to total β-catenin protein 3.3-fold

(p=0.007; Figure 5B).

Growth signaling. Exercise had no effect on polyp Akt phosphorylation protein levels (1.00 ±

0.11 vs. 0.88 ± 0.10 normalized IOD), total protein levels (1.00 ± 0.06 vs. 1.07 ± 0.04

normalized IOD), or the ratio of phosphorylated to total Akt protein in polyps (Figures 5A &

5B).

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DISCUSSION

Many studies have examined the effect of exercise on ApcMin/+ mouse intestinal polyp

burden (7-9, 28). To our knowledge, this is the first study to examine the effect of exercise on

ApcMin/+ mouse polyp cellular characteristics and protein expression. We report that exercise

reduced the number of macrophages in intestinal polyps from ApcMin/+ mice, but the number of

COX-2 positive cells remained unchanged by exercise. It appears cells other than macrophages

are also sources of COX-2 signaling in polyps. Further work can establish if the beneficial effect

of exercise on polyp number and growth is independent of COX-2 signaling. We also found that

exercise decreased markers of apoptosis and increased β-catenin phosphorylation in polyps.

Together, these findings suggest that exercise can influence several signaling pathways related to

polyp formation and growth.

One potential exercise-mediated mechanism of colorectal cancer prevention is its anti-

inflammatory effect (36, 51). Our lab has previously published that exercise trained ApcMin/+

mice have a reduction in circulating IL-6 levels, which is associated with a decrease in polyp

number and size (28). This decrease in polyp burden was also correlated with a reduction in the

crypt depth:villus height ratio, and indirect marker of intestinal inflammation. In other cancer

models, daily strenuous exercise can reduce tumor macrophage and neutrophil infiltration, which

is associated with delayed tumor growth (53). While macrophages are typically thought to fight

tumors, tumor-associated macrophages secrete inflammatory proteins that contribute to tumor

growth and attract more inflammatory cells (16, 22). In the current study, exercise decreased the

number of macrophages in polyps, and the effect was more pronounced in large polyps. Since

these effects were only found in large polyps, these data suggest that the exercise-induced

reduction in ApcMin/+ mouse polyp growth is partially attributed to a reduction in the polyp

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inflammatory state. COX-2-secreting cells in ApcMin/+ polyps are macrophages (16). The role of

COX-2 in tumor progression is the secretion of prostaglandins to stimulate growth. Since COX-

2-positive cells and protein expression were unaltered in polyps, exercise’s effect on macrophage

polyp formation may not be mediated through reduced COX-2 expression. Therefore, exercise

has the potential to block polyp growth by decreasing the polyp inflammatory environment,

through immune cell recruitment.

Counteracting cell growth and replication is apoptosis. A common characteristic of

tumor cells is that they lack the ability to carry out apoptosis (12). Many ApcMin/+ mouse studies

that detect a reduction in tumor burden also detect increases in apoptosis with these same

treatments (41, 52). In the current study, small polyps from exercised mice had fewer TUNEL-

positive cells. This was accompanied by a decrease in polyp Bax protein expression, a pro-

apoptotic protein. Since this running protocol was associated with a decrease in polyp number

and size, these findings were unexpected. However, when examining wild-type and ApcMin/+

mouse intestinal gene expression, apoptotic genes are up-regulated in adenomas compared to

normal epithelia (35). This appears to be a function of Apc since conditional Apc deletion within

the intestine or neural crest cells increases apoptosis (13, 43). ApcMin/+ mice are heterozygotes

for the Apc gene, loss of heterozygosity within individual cells leads to polyp formation. Since

exercised mice have fewer apoptotic cells in their polyps, this may be a reflection of Apc

heterozygosity. These data suggest that exercised mice may have more polyp cells that are

heterozygous for Apc and subsequently, less apoptosis. Further examination of this should be

verified by loss of heterozygosity assays (LOH) in sedentary and exercised ApcMin/+ mouse

intestinal polyps. In addition, exercise reduced apoptosis only in small polyps, but not in large

polyps. These data suggest that exercise-induced decreases in ApcMin/+ mouse polyp formation

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may be mediated through apoptosis. The lack of a change in apoptosis in large polyps suggests

that the exercise-induced reduction in ApcMin/+ mouse polyp growth does not appear to be due to

an apoptotic mechanism.

The loss of Apc leads to apoptosis and to β-catenin nuclear localization, when turns on

proliferation genes, such as c-myc and cyclin D1 (4, 38). β-catenin expression is prominent in

ApcMin/+ mouse intestinal polyps (17, 20, 21, 29) and precancerous lesions also have abundant β-

catenin expression (15). Therapeutics that decrease intestinal tumor burden are associated with

changes in β-catenin content and/or localization (20). Immunohistochemsitry revealed intense

nuclear β-catenin staining of the crypts within polyps. However, exercise did not change the

number of β-catenin positive crypts within polyps. However, β-catenin phosphorylation was

increased with exercise. Since β-catenin is downstream from Apc, and Apc loss leads to polyp

formation, these data would suggest that exercise modulates the Apc→β-catenin pathway.

Further work is needed to determine the downstream targets of β-catenin signaling, as well as β-

catenin localization, that are modulated within ApcMin/+ mouse intestinal polyps following

exercise training.

Circulating factors, such as insulin or IGF-I, can promote tumorgenesis and growth via

the PI3K/Akt pathway (11, 14). A reduction in plasma insulin or IGF-I has been hypothesized as

a possible exercise-induced mechanism of cancer prevention (3, 24, 32, 33, 39). Akt activity is

increased in ApcMin/+ intestinal polyps and a reduction in polyp burden is associated with

inhibition of this signaling pathway (20, 30, 35). In the present study, phosphorylated and total

Akt levels were not different in intestinal polyps between sedentary and exercise-trained ApcMin/+

mice. While other proteins can influence Akt activity, these data coincide with previous ApcMin/+

mouse exercise studies that do not detect a reduction in circulating insulin or IGF-I levels,

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despite a reduction in tumor number (8, 9, 28). In summary, this study demonstrates that

exercise can regulate ApcMin/+ mouse intestinal polyp composition. The effects of exercise that

reduce the overall tumor burden (size & number) in the ApcMin/+ mouse likely occur via multiple

cellular mechanisms, including reduced immune cell infiltration, apoptosis, and β-catenin

signaling.

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ACKNOWLEDGEMENTS

The authors would like to thank Tia Davis and Valerie Kennedy for technical assistance.

The research described in this report was supported by NIH Grant P20 RR-017698 from the

National Center for Research Resources. Its contents are solely the responsibility of the authors

and do not necessarily represent the official views of the NIH.

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FIGURE LEGENDS

Figure 1. Macrophages in intestinal polyps following 9 weeks of exercise training. F4/80

positive cells were counted in polyps located in the distal ileum of male ApcMin/+ mice after 9

weeks of treadmill training. A. Control polyp (x400 magnification). B. Exercise polyp (x400

magnification). C. F4/80 data. Data are means ± SE. Data were analyzed with independent t-

tests. Significance was set at p<0.05. *Signifies different from Control.

Figure 2. COX-2 positive cells were counted in polyps located in the distal ileum of male

ApcMin/+ mice after 9 weeks of treadmill training. A. Control polyp (x400 magnification). B.

Exercise polyp (x400 magnification). C. COX-2 data. Data are means ± SE. Data were

analyzed with independent t-tests. Significance was set at p<0.05. *Signifies different from

Control.

Figure 3. Apoptotic cells in polyps following exercise training. TUNEL-positive cells were

counted in polyps located in the distal ileum of male ApcMin/+ mice after 9 weeks of treadmill

training. A. Control polyp (x200 magnification). B. Exercise polyp (x200 magnification). C.

TUNEL data. Data are means ± SE. Data were analyzed with independent t-tests. Significance

was set at p<0.05. *Signifies different from Control.

Figure 4. β-Catenin staining in polyps following exercise training. β-Catenin foci were counted

in polyps located in the distal ileum of male ApcMin/+ mice after 9 weeks of treadmill training.

Polyps were categorized as having a small number of foci (<5 foci) or a large number of foci (>5

foci). A. Control polyp (x200 magnification). B. Exercise polyp (x200 magnification). C. β-

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Catenin data. Data are means ± SE. Data were analyzed with independent t-tests within each

polyp size category. Significance was set at p<0.05.

Figure 5. Protein expression in ApcMin/+ mouse intestinal polyps. A. Representative Western

blot and Ponceau stain of COX-2, Bax, β-catenin, and Akt protein expression in intestinal

polyps. B. Representative means and SE of each protein. β-Catenin and AKT data represent the

ratios of the phosphorylated forms to the total amount. The IOD values are normalized to

Control mice. Values are means ± SE. Data were analyzed with independent t-tests.

Significance was set at p<0.05. *Signifies different from Control.

Page 26 of 31

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Figure 4. Page 30 of 31

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