Carcinogenesis AdvanceCarcinogenesis Access published December 30, 2014
Cholera-toxin suppresses carcinogenesis in a mouse model of inflammation-driven sporadic colon cancer
CARCIN-2014-00696.R2
Fo
Manuscript ID:
Carcinogenesis
Manuscript Type: Date Submitted by the Author:
Original Manuscript
09-Dec-2014
rP
Complete List of Authors:
Keywords:
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Poutahidis, Theofilos; Aristotle University of Thessaloniki, Laboratory of Pathology, Faculty of Health Sciences, School of Veterinary Medicine Doulberis, Michael; Aristotle University of Thessaloniki, Laboratory of Pathology, Faculty of Health Sciences, School of Veterinary Medicine Angelopoulou, Katerina; Aristotle University of Thessaloniki, Laboratory of Biochemistry and Toxicology, Faculty of Health Sciences, School of Veterinary Medicine Kaldrymidou, Eleni; Aristotle University of Thessaloniki, Laboratory of Pathology, Faculty of Health Sciences, School of Veterinary Medicine Tsingotjidou, Anastasia; Aristotle University of Thessaloniki, Laboratory of Anatomy, Histology and Embryology, Faculty of Health Sciences, School of Veterinary Medicine Abas, Zaphiris; Democritus University of Thrace, Department of Agricultural Development Erdman, Susan; Massachusetts Institute of Technology, Division of Comparative Medicine
tumor microenvironment, colon cancer, cholera-toxin, inflammation, mice
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Downloaded from http://carcin.oxfordjournals.org/ at University of California, San Diego on January 16, 2015
Journal:
Page 1 of 41
Carcinogenesis
1 1
Cholera-toxin suppresses carcinogenesis in a mouse model of inflammation-driven sporadic
2
colon cancer
3 4
Michael Doulberis, Katerina Angelopoulou1, Eleni Kaldrymidou, Anastasia Tsingotjidou2, Zaphiris
5
Abas3†, Suzan E. Erdman4 and Theofilos Poutahidis*
6 7
Laboratory of Pathology, 1Laboratory of Biochemistry and Toxicology, 2Laboratory of Anatomy,
8
Histology and Embryology, Faculty of Health Sciences, School of Veterinary Medicine, Aristotle
9
University of Thessaloniki, Thessaloniki, 54124, Greece; 3Department of Agricultural Development,
10
Democritus University of Thrace, Orestiada, 68200, Greece; 4Division of Comparative Medicine,
11
Massachusetts Institute of Technology, Cambridge MA, 02139, USA
ee
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Fo
12 13
Running Title: CT reduces colonic adenomas
14
Keywords: tumor microenvironment, colon cancer, cholera-toxin, inflammation, mice
15
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
16
*To whom correspondence should be addressed. Tel: +302310999810; Fax: +302310999979. E-
17
mail: [email protected]
18 19
† 1966-2013; This paper is dedicated to his memory.
20 21
Abbreviations:
22
AOM: azoxymethane; CIS: carcinoma in situ; CRC: colorectal cancer; CT: cholera-toxin; DSS: dextran
23
sodium sulfate; HGD: high grade dysplasia; IHC: immunohistochemistry; IL: interleukin; LGD: low
24
grade dysplasia; MLN: mesenteric lymph nodes; MPO: myeloperoxidase; Treg: Regulatory T-cells
25
Carcinogenesis
Page 2 of 41
2 26
Abstract
27
Human studies and clues from animal models have provided important links between
28
gastrointestinal (GI) tract bacteria and colon cancer. Gut microbiota antigenic stimuli play an
29
important role in shaping the intestinal immune responses. Therefore, especially in the case of
30
inflammation-associated colon cancer, gut bacteria antigens may affect tumorigenesis. The present
31
study aimed to investigate the effects of the oral administration of a bacterial product with known
32
immunomodulatory properties on inflammation-driven colorectal neoplasmatogenesis. For that,
33
we used cholera-toxin and a well-established mouse model of colon cancer in which neoplasia is
34
initiated by a single dose of the genotoxic agent azoxymethane and subsequently promoted by
35
inflammation caused by the colitogenic substance dextran sodium sulfate. We found that a single,
36
low, non-pathogenic dose of cholera-toxin, given orally at the beginning of each dextran sodium
37
sulfate treatment cycle downregulated neutrophils and upregulated regulatory T-cells and IL-10 in
38
the colonic mucosa. The cholera-toxin-induced disruption of the tumor-promoting character of
39
dextran sodium sulfate-induced inflammation led to the reduction of the azoxymethane-initiated
40
colonic polypoidogenesis. This result adds value to the emerging notion that certain
41
gastrointestinal tract bacteria or their products affect the immune system and render the
42
microenvironment of preneoplastic lesions less favorable for promoting their evolution to cancer.
43
(word count=202)
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
44 45
Summary
46
The present study shows that cholera-toxin suppresses azoxymethane-initiated, dextran sodium
47
sulfate-promoted colonic polypoidogenesis in mice. The oral administration of cholera-toxin
48
disrupts the tumor-promoting character of colitis, by downregulating neutrophils and upregulating
49
regulatory T-cells and IL-10 in the colonic mucosa.
50
(word count=39)
Page 3 of 41
Carcinogenesis
3 51 52
Introduction
53
The prevalence of certain neoplasms, including colorectal cancer, is alarmingly high in the
54
industrialized world[1]. In recent years, human studies and clues from animal models have
55
provided important links between gastrointestinal (GI) tract bacteria and colon cancer[2,3]. Gut
56
microbiota antigenic stimuli play an important role in shaping the intestinal immune responses[4].
57
Therefore, at least in the case of inflammation-associated colon cancer, gut bacteria antigens may
58
influence carcinogenesis[5-7].
59
Fo
60
In a series of studies in mice we have shown that some gastrointestinal (GI) tract bacteria induce
61
regulatory T-cells (Treg) of increased anti-inflammatory potency, which in turn counteract epithelial
62
tumors throughout the body, instead of promoting them through anti-tumor immunity
63
downregulation [6,8-11]. Importantly, the bacteria-primed Treg activate clinically silent local GI
64
tract and systematic immunity networks, which confer decreased risk of epithelial
65
carcinogenesis[10,11]. These findings are in line with the recognized importance of immune cells
66
and factors in tumor evolution and their emergence as key elements of the tumor micro- and
67
macro-environment[12]. Further, they suggest that tumor survival and progression may be more
68
vitally dependent on elevated and sustained local and systematic proinflammatory signaling than
69
previously thought[6,12].
w ie ev
rR
ee
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
70 71
Following this reasoning, the use of selected bacterial antigens to modulate cancer-promoting
72
inflammatory events is worth exploring[2,5,6]. These products may be a safe and effective
73
approach for reinforcing the regulatory arm of the human immune system. Ideally, they may
74
contribute to the decrease of the modern living-associated uncontrolled inflammation and cancer
75
risk at the population level[6,8,13].
Carcinogenesis
Page 4 of 41
4 76 77
The small intestinal pathogenic bacterium Vibrio cholerae is the causative agent of cholera. The
78
developing world is not endemic, however, cholera is endemic in the developing world[14]. The
79
major pathogenic factor of Vibrio cholerae is cholera-toxin (CT), a protein exotoxin responsible for
80
the life-threatening massive diarrhea, which is the main clinical presentation of cholera[14]. CT's
81
mode of pathogenic action has been well characterized[14,15]. The bulk of research now focuses
82
more on its powerful immune response adjuvant and immunomodulating properties[15,16]. Orally
83
introduced CT in low, non-pathogenic doses alters gut mucosal immunity and effectively enhances
84
the induction of antigen-specific oral tolerance[15-17]. For that, CT is considered an attractive
85
component of oral tolerance-based immunotherapy schemes for the treatment of immune-related
86
disorders[15-17]. CT activates antigen-presenting cells[16,18-21], induces Tregs and expands their
87
population[16,17,19], affects the expression of cytokines and modulates both Th1 and Th2
88
immune responses[16,17,20,22]. Interestingly, these immune events have been shown to affect
89
carcinogenic processes[12].
w ie ev
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ee
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Fo
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
90 91
In the present study, we sought to examine the effects of CT oral administration on colorectal (CRC)
92
neoplasmatogenesis. For that, we used a well-established mouse model of CRC in which cancer is
93
initiated by a single dose of the genotoxic agent azoxymethane (AOM) and subsequently promoted
94
by inflammation caused by the colitogenic substance dextran sodium sulfate (DSS)[23]. We found
95
that by downregulating neutrophils and upregulating regulatory T-cells and IL-10, CT alters the
96
inflammatory milieu of cancer-promoting colitis and decreases the risk of CRC.
97 98 99 100 101 102
Materials and Methods Animals Specific pathogen-free certified BALB/cJ mice were purchased from Jackson Laboratories (Bar
Page 5 of 41
Carcinogenesis
5 103
Harbor, ME). Mice were kept bio-contained in static micro-isolator cages, fed with sterilized regular
104
mouse chow, and given sterilized water. Their helicobacter-free status was confirmed by
105
polymerase chain reaction (PCR) as previously described[24]. Animal experiments were approved
106
by the Faculty of Veterinary Medicine, Aristotle University of Thessaloniki and licensed by the
107
National Veterinary Administration authorities (License No. 13/10621/11.08.2008).
108 109 110 111
Experimental Design
112
injection of the carcinogen AOM (10mg/kg of body weight). One week later, 1% DSS (molecular
113
weight: 36-50 kDa; MP Biomedicals Inc, Cleveland, OH) was given in the drinking water for 1 week
114
followed by 1 week of regular water. This cycle was repeated three times. At the first day of each
115
cycle of DSS exposure, 10 μg of CT were inoculated by gastric gavage. Mice were either treated
116
with DSS, AOM and CT in all possible combinations or remained untreated. Mice were killed either
117
at 3.5 months (first experiment—long term) or at 3 days (second experiment—short term) after
118
the last week of DSS exposure. Numbers of mice per experimental group for each experiment were
119
as follows: first experiment: Control (n=10), CT (n=10), DSS (n=10), AOM (n=10), DSS-CT (n=10),
120
AOM-CT (n=10), AOM-DSS (n=10), AOM-DSS-CT (n=10); second experiment: AOM-DSS (n=5), AOM-
121
DSS-CT (n=5).
122 123
Necropsy and Tissue Sampling
124
Mice were killed with an overdose of Isofluorane, weighted and necropsied. The colon of mice was
125
removed, cut-open and photographed in high resolution for grossly visible polyp counts. For gene
126
expression analysis 1 cm colon samples were collected from a standard area of the proximal part of
127
descending colon, immediately immersed in an RNA-later solution (Takara Bio Inc., Shiga, Japan)
128
and stored at -80o C until further processing. The remaining colon was fixed in 10% neutral-
A total of 90 male mice were used. At the age of 5-6 weeks mice were injected with a single i.p.
w ie ev
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ee
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Fo
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Carcinogenesis
Page 6 of 41
6 129
buffered formalin for histological and immunohistochemical analyses.
130 131
Histopathology, Immunohistochemistry and Morphometry
132
For histologic evaluation, formalin-fixed colon and mesenteric lymph nodes (MLN) were embedded
133
in paraffin, cut at 5 μm, and stained with hematoxylin and eosin or immunohistochemistry (IHC).
134
Dysplastic and neoplastic lesions and mucosal/submucosal inflammation in the colon were scored
135
on 0–4 ascending scales using criteria which have been previously described in detail[24,25].
136
Primary antibodies for IHC included a) rabbit polyclonal antibodies against β-catenin,
137
myeloperoxidase (ThermoFisher Scientific/Lab Vision, Fremont, CA), E-cadherin, IL-17, Tgfβ-1
138
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA), cleaved caspase-3 (Cell Signaling, Beverly, MA) and
139
CD3 (Cell Marque, Rocklin, CA); b) rabbit monoclonal antibodies against Ki-67 and c-kit (Cell
140
Marque); c) rat monoclonal antibodies against Foxp3 (eBioscience, Inc., San Diego, CA) and F4/80
141
(Serotec, Oxford, UK); and d) a goat polyclonal antibody against interleukin (IL)-16 (Santa Cruz
142
Biotechnology, Inc.). IHC and quantitative histomorphometry were performed as described
143
previously[24].
144
w ie ev
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ee
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Fo
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
145
Quantitative Gene Expression Analysis
146
Real-time PCR based on the SYBR Green chemistry was used to quantitatively analyze the
147
expression of Tumor Necrosis Factor (TNF)-α, IL-6, IL-10 and Transforming growth factor (Tgf)-β1,
148
as previously described[24].
149 150
Statistical analyses
151
Adenomatous polyp counts were analyzed by unpaired Student’s t test with Welch's correction.
152
Histomorphomety and relative gene expression data were compared between groups using
153
Kruskal-Wallis one-way analysis of variance and Dunn's post-test or Mann–Whitney U analysis.
Page 7 of 41
Carcinogenesis
7 154
Statistical significance was set at P
Cholera-toxin suppresses carcinogenesis in a mouse model of inflammation-driven sporadic colon cancer
CARCIN-2014-00696.R2
Fo
Manuscript ID:
Carcinogenesis
Manuscript Type: Date Submitted by the Author:
Original Manuscript
09-Dec-2014
rP
Complete List of Authors:
Keywords:
ie ev
rR
ee
Poutahidis, Theofilos; Aristotle University of Thessaloniki, Laboratory of Pathology, Faculty of Health Sciences, School of Veterinary Medicine Doulberis, Michael; Aristotle University of Thessaloniki, Laboratory of Pathology, Faculty of Health Sciences, School of Veterinary Medicine Angelopoulou, Katerina; Aristotle University of Thessaloniki, Laboratory of Biochemistry and Toxicology, Faculty of Health Sciences, School of Veterinary Medicine Kaldrymidou, Eleni; Aristotle University of Thessaloniki, Laboratory of Pathology, Faculty of Health Sciences, School of Veterinary Medicine Tsingotjidou, Anastasia; Aristotle University of Thessaloniki, Laboratory of Anatomy, Histology and Embryology, Faculty of Health Sciences, School of Veterinary Medicine Abas, Zaphiris; Democritus University of Thrace, Department of Agricultural Development Erdman, Susan; Massachusetts Institute of Technology, Division of Comparative Medicine
tumor microenvironment, colon cancer, cholera-toxin, inflammation, mice
w
Downloaded from http://carcin.oxfordjournals.org/ at University of California, San Diego on January 16, 2015
Journal:
Page 1 of 41
Carcinogenesis
1 1
Cholera-toxin suppresses carcinogenesis in a mouse model of inflammation-driven sporadic
2
colon cancer
3 4
Michael Doulberis, Katerina Angelopoulou1, Eleni Kaldrymidou, Anastasia Tsingotjidou2, Zaphiris
5
Abas3†, Suzan E. Erdman4 and Theofilos Poutahidis*
6 7
Laboratory of Pathology, 1Laboratory of Biochemistry and Toxicology, 2Laboratory of Anatomy,
8
Histology and Embryology, Faculty of Health Sciences, School of Veterinary Medicine, Aristotle
9
University of Thessaloniki, Thessaloniki, 54124, Greece; 3Department of Agricultural Development,
10
Democritus University of Thrace, Orestiada, 68200, Greece; 4Division of Comparative Medicine,
11
Massachusetts Institute of Technology, Cambridge MA, 02139, USA
ee
rP
Fo
12 13
Running Title: CT reduces colonic adenomas
14
Keywords: tumor microenvironment, colon cancer, cholera-toxin, inflammation, mice
15
w ie ev
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
16
*To whom correspondence should be addressed. Tel: +302310999810; Fax: +302310999979. E-
17
mail: [email protected]
18 19
† 1966-2013; This paper is dedicated to his memory.
20 21
Abbreviations:
22
AOM: azoxymethane; CIS: carcinoma in situ; CRC: colorectal cancer; CT: cholera-toxin; DSS: dextran
23
sodium sulfate; HGD: high grade dysplasia; IHC: immunohistochemistry; IL: interleukin; LGD: low
24
grade dysplasia; MLN: mesenteric lymph nodes; MPO: myeloperoxidase; Treg: Regulatory T-cells
25
Carcinogenesis
Page 2 of 41
2 26
Abstract
27
Human studies and clues from animal models have provided important links between
28
gastrointestinal (GI) tract bacteria and colon cancer. Gut microbiota antigenic stimuli play an
29
important role in shaping the intestinal immune responses. Therefore, especially in the case of
30
inflammation-associated colon cancer, gut bacteria antigens may affect tumorigenesis. The present
31
study aimed to investigate the effects of the oral administration of a bacterial product with known
32
immunomodulatory properties on inflammation-driven colorectal neoplasmatogenesis. For that,
33
we used cholera-toxin and a well-established mouse model of colon cancer in which neoplasia is
34
initiated by a single dose of the genotoxic agent azoxymethane and subsequently promoted by
35
inflammation caused by the colitogenic substance dextran sodium sulfate. We found that a single,
36
low, non-pathogenic dose of cholera-toxin, given orally at the beginning of each dextran sodium
37
sulfate treatment cycle downregulated neutrophils and upregulated regulatory T-cells and IL-10 in
38
the colonic mucosa. The cholera-toxin-induced disruption of the tumor-promoting character of
39
dextran sodium sulfate-induced inflammation led to the reduction of the azoxymethane-initiated
40
colonic polypoidogenesis. This result adds value to the emerging notion that certain
41
gastrointestinal tract bacteria or their products affect the immune system and render the
42
microenvironment of preneoplastic lesions less favorable for promoting their evolution to cancer.
43
(word count=202)
w ie ev
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ee
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
44 45
Summary
46
The present study shows that cholera-toxin suppresses azoxymethane-initiated, dextran sodium
47
sulfate-promoted colonic polypoidogenesis in mice. The oral administration of cholera-toxin
48
disrupts the tumor-promoting character of colitis, by downregulating neutrophils and upregulating
49
regulatory T-cells and IL-10 in the colonic mucosa.
50
(word count=39)
Page 3 of 41
Carcinogenesis
3 51 52
Introduction
53
The prevalence of certain neoplasms, including colorectal cancer, is alarmingly high in the
54
industrialized world[1]. In recent years, human studies and clues from animal models have
55
provided important links between gastrointestinal (GI) tract bacteria and colon cancer[2,3]. Gut
56
microbiota antigenic stimuli play an important role in shaping the intestinal immune responses[4].
57
Therefore, at least in the case of inflammation-associated colon cancer, gut bacteria antigens may
58
influence carcinogenesis[5-7].
59
Fo
60
In a series of studies in mice we have shown that some gastrointestinal (GI) tract bacteria induce
61
regulatory T-cells (Treg) of increased anti-inflammatory potency, which in turn counteract epithelial
62
tumors throughout the body, instead of promoting them through anti-tumor immunity
63
downregulation [6,8-11]. Importantly, the bacteria-primed Treg activate clinically silent local GI
64
tract and systematic immunity networks, which confer decreased risk of epithelial
65
carcinogenesis[10,11]. These findings are in line with the recognized importance of immune cells
66
and factors in tumor evolution and their emergence as key elements of the tumor micro- and
67
macro-environment[12]. Further, they suggest that tumor survival and progression may be more
68
vitally dependent on elevated and sustained local and systematic proinflammatory signaling than
69
previously thought[6,12].
w ie ev
rR
ee
rP
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
70 71
Following this reasoning, the use of selected bacterial antigens to modulate cancer-promoting
72
inflammatory events is worth exploring[2,5,6]. These products may be a safe and effective
73
approach for reinforcing the regulatory arm of the human immune system. Ideally, they may
74
contribute to the decrease of the modern living-associated uncontrolled inflammation and cancer
75
risk at the population level[6,8,13].
Carcinogenesis
Page 4 of 41
4 76 77
The small intestinal pathogenic bacterium Vibrio cholerae is the causative agent of cholera. The
78
developing world is not endemic, however, cholera is endemic in the developing world[14]. The
79
major pathogenic factor of Vibrio cholerae is cholera-toxin (CT), a protein exotoxin responsible for
80
the life-threatening massive diarrhea, which is the main clinical presentation of cholera[14]. CT's
81
mode of pathogenic action has been well characterized[14,15]. The bulk of research now focuses
82
more on its powerful immune response adjuvant and immunomodulating properties[15,16]. Orally
83
introduced CT in low, non-pathogenic doses alters gut mucosal immunity and effectively enhances
84
the induction of antigen-specific oral tolerance[15-17]. For that, CT is considered an attractive
85
component of oral tolerance-based immunotherapy schemes for the treatment of immune-related
86
disorders[15-17]. CT activates antigen-presenting cells[16,18-21], induces Tregs and expands their
87
population[16,17,19], affects the expression of cytokines and modulates both Th1 and Th2
88
immune responses[16,17,20,22]. Interestingly, these immune events have been shown to affect
89
carcinogenic processes[12].
w ie ev
rR
ee
rP
Fo
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
90 91
In the present study, we sought to examine the effects of CT oral administration on colorectal (CRC)
92
neoplasmatogenesis. For that, we used a well-established mouse model of CRC in which cancer is
93
initiated by a single dose of the genotoxic agent azoxymethane (AOM) and subsequently promoted
94
by inflammation caused by the colitogenic substance dextran sodium sulfate (DSS)[23]. We found
95
that by downregulating neutrophils and upregulating regulatory T-cells and IL-10, CT alters the
96
inflammatory milieu of cancer-promoting colitis and decreases the risk of CRC.
97 98 99 100 101 102
Materials and Methods Animals Specific pathogen-free certified BALB/cJ mice were purchased from Jackson Laboratories (Bar
Page 5 of 41
Carcinogenesis
5 103
Harbor, ME). Mice were kept bio-contained in static micro-isolator cages, fed with sterilized regular
104
mouse chow, and given sterilized water. Their helicobacter-free status was confirmed by
105
polymerase chain reaction (PCR) as previously described[24]. Animal experiments were approved
106
by the Faculty of Veterinary Medicine, Aristotle University of Thessaloniki and licensed by the
107
National Veterinary Administration authorities (License No. 13/10621/11.08.2008).
108 109 110 111
Experimental Design
112
injection of the carcinogen AOM (10mg/kg of body weight). One week later, 1% DSS (molecular
113
weight: 36-50 kDa; MP Biomedicals Inc, Cleveland, OH) was given in the drinking water for 1 week
114
followed by 1 week of regular water. This cycle was repeated three times. At the first day of each
115
cycle of DSS exposure, 10 μg of CT were inoculated by gastric gavage. Mice were either treated
116
with DSS, AOM and CT in all possible combinations or remained untreated. Mice were killed either
117
at 3.5 months (first experiment—long term) or at 3 days (second experiment—short term) after
118
the last week of DSS exposure. Numbers of mice per experimental group for each experiment were
119
as follows: first experiment: Control (n=10), CT (n=10), DSS (n=10), AOM (n=10), DSS-CT (n=10),
120
AOM-CT (n=10), AOM-DSS (n=10), AOM-DSS-CT (n=10); second experiment: AOM-DSS (n=5), AOM-
121
DSS-CT (n=5).
122 123
Necropsy and Tissue Sampling
124
Mice were killed with an overdose of Isofluorane, weighted and necropsied. The colon of mice was
125
removed, cut-open and photographed in high resolution for grossly visible polyp counts. For gene
126
expression analysis 1 cm colon samples were collected from a standard area of the proximal part of
127
descending colon, immediately immersed in an RNA-later solution (Takara Bio Inc., Shiga, Japan)
128
and stored at -80o C until further processing. The remaining colon was fixed in 10% neutral-
A total of 90 male mice were used. At the age of 5-6 weeks mice were injected with a single i.p.
w ie ev
rR
ee
rP
Fo
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Carcinogenesis
Page 6 of 41
6 129
buffered formalin for histological and immunohistochemical analyses.
130 131
Histopathology, Immunohistochemistry and Morphometry
132
For histologic evaluation, formalin-fixed colon and mesenteric lymph nodes (MLN) were embedded
133
in paraffin, cut at 5 μm, and stained with hematoxylin and eosin or immunohistochemistry (IHC).
134
Dysplastic and neoplastic lesions and mucosal/submucosal inflammation in the colon were scored
135
on 0–4 ascending scales using criteria which have been previously described in detail[24,25].
136
Primary antibodies for IHC included a) rabbit polyclonal antibodies against β-catenin,
137
myeloperoxidase (ThermoFisher Scientific/Lab Vision, Fremont, CA), E-cadherin, IL-17, Tgfβ-1
138
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA), cleaved caspase-3 (Cell Signaling, Beverly, MA) and
139
CD3 (Cell Marque, Rocklin, CA); b) rabbit monoclonal antibodies against Ki-67 and c-kit (Cell
140
Marque); c) rat monoclonal antibodies against Foxp3 (eBioscience, Inc., San Diego, CA) and F4/80
141
(Serotec, Oxford, UK); and d) a goat polyclonal antibody against interleukin (IL)-16 (Santa Cruz
142
Biotechnology, Inc.). IHC and quantitative histomorphometry were performed as described
143
previously[24].
144
w ie ev
rR
ee
rP
Fo
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
145
Quantitative Gene Expression Analysis
146
Real-time PCR based on the SYBR Green chemistry was used to quantitatively analyze the
147
expression of Tumor Necrosis Factor (TNF)-α, IL-6, IL-10 and Transforming growth factor (Tgf)-β1,
148
as previously described[24].
149 150
Statistical analyses
151
Adenomatous polyp counts were analyzed by unpaired Student’s t test with Welch's correction.
152
Histomorphomety and relative gene expression data were compared between groups using
153
Kruskal-Wallis one-way analysis of variance and Dunn's post-test or Mann–Whitney U analysis.
Page 7 of 41
Carcinogenesis
7 154
Statistical significance was set at P
