Enhancement of development of azoxymethane-induced colonic premalignant lesions in C57BL/KsJ-db/db mice
Yoshinobu Hirose3,
Kazuya Hata,
Toshiya Kuno,
Koujiro Yoshida,
Keiko Sakata,
Yasuhiro Yamada,
Takuji Tanaka1,
Bandaru S. Reddy2 and
Hideki Mori
Department of Tumor Pathology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu 500-8705, Japan, 1 Department of Pathology, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan and 2 Institute for Cancer Prevention, 1 Dana Road, Valhalla, NY 10595, USA
3 To whom correspondence should be addressed Email: yhirose{at}cc.gifu-u.ac.jp
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Abstract
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Epidemiological studies have shown that obesity and diabetes mellitus may be risk factors for colon cancer. However, the underlying mechanisms of how these chronic diseases promote colon carcinogenesis remain unknown. C57BL/KsJ-db/db mice have obese and diabetic phenotypes because of disruption of the leptin receptor. The present study was designed to investigate whether development of azoxymethane (AOM)-induced dysplastic and early neoplastic (premalignant) lesions of the colon is modulated in db/db mice. Homozygous db/db mice, heterozygous db/+ mice and littermate controls (+/+) were injected with AOM under food restriction (
10.8 kcal/mouse/day) and killed 5 weeks after the carcinogen treatment. Their colons were assessed for premalignant lesions induced by AOM. We found a significant increase in the multiplicity of the total premalignant lesions in db/db mice when compared with db/+ or +/+ mice. Phenotypically, serum leptin and insulin levels in db/db mice were significantly higher than those in db/+ or +/+ mice, whereas the body weights and glucose levels in blood of db/db, db/+ and +/+ mice were comparable. In addition, immunostaining of the leptin receptor and insulin-like growth factor-I receptor showed up-regulation of these protein levels specifically in the lesions. Our data indicate that development of AOM-induced premalignant lesions is enhanced in db/db mice with hyperleptinemia and hyperinsulinemia. The results have important implications for further exploration of the possible underlying events that affect the positive association between colon cancer and chronic diseases (obesity and diabetes).
Abbreviations: ACF, aberrant crypt foci; AOM, azoxymethane; EIA, enzyme immunoassay; H&E, hematoxylin and eosin; IGF-I, insulin-like growth factor-I; IGF-IR, insulin-like growth factor-I receptor; MAPK, mitogen-activated protein kinase; Ob-R, leptin receptor
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Introduction
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Numerous epidemiological results suggest that obesity is a risk factor for colon cancer (1,2). In spite of the accumulating evidence in epidemiology, experimental and laboratory studies to confirm the aforementioned proposal and reveal the underlying mechanisms have not been extensively performed so far. This may be because obesity is a complex, heterogeneous and multifactorial syndrome resulting from both genetic susceptibility and environmental factors (3). Besides obesity, it is well known that several factors, including a high fat and low fiber diet (4), low physical activity (5), inflammatory bowel diseases (6) or hereditary disorders such as familial adenomatous polyposis and non-polyposis syndrome (7), increase the risk for development of colorectal cancers. Interestingly, some of the risk factors for colon cancer, such as a high fat diet or decreased physical activity, enhance obesity as well (8,9), suggesting that there might be common biological events or undefined interactive events that affect the positive association between colon cancer and weight gain. Identification and evaluation of such phenomena will be critical in the near future for preventive strategies against both diseases.
C57BL/KsJ-db/db mice are genetically altered models with phenotypes of obesity and diabetes mellitus (10). Disruption of the leptin receptor (Ob-R) gene in these mice leads to overexpression of leptin in the adipose tissue and a concomitantly high concentration of leptin in the blood of the mice (11,12). It is widely accepted that leptin functions as a satiety factor through Ob-R, which is mainly expressed in the hypothalamus (12). Because of a deficiency of the leptin-mediated satiety signaling, abnormal dietary habits such as hyperphagia occur in homozygous db/db mice, resulting in complex phenotypes. It is now well established that leptin not only interacts with pathways in the central nervous system, but also functions in the peripheral tissues as a mediator of energy expenditure, a permissive factor for puberty and a signal of metabolic status (13). Interestingly, some lines of evidence suggest that leptin in the periphery behaves as a growth factor in lung (14), breast (15) and colonic tissues (16). A plausible role of leptin in tumorigenesis remains undetermined, although there have been several studies suggesting the leptin-related pathway as a possible modulator in neoplastic development (1719).
In the present study we conducted a short-term assay to examine whether occurrence of azoxymethane (AOM)-induced dysplastic and early neoplastic lesions of colon is modulated in genetically obese db/db mice. The main goal of this study was to assess the involvement of obesity-related events such as hyperleptinemia in colon carcinogenesis in vivo. To do so, the involvement of dietary factors, including hyperphagia, was kept minimal in this experiment because these may dominate or mask obesity-related interactive factors. To this end, food intake by all the experimental mice was moderately and equally restricted during the study.
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Materials and methods
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Animals, diets, and carcinogen
A total of 50 animals (15 male 4-week-old C57BL/KsJ-db/db mice, 18 male 4-week-old C57BL/KsJ-db/+ mice and 17 male 4-week-old C57BL/KsJ-+/+ mice), purchased from Jackson Laboratories (Bar Harbor, ME), were used. The mice were housed in a holding room under controlled conditions of a 12 h light/dark cycle, 23 ± 2°C room temperature and 50 ± 10% relative humidity. From 4 weeks of age they were randomly assigned to experimental (1013 mice of each genotype) and control (5 mice each) groups and were housed in small cages separately to control the food intake of all animals. MF (Oriental Yeast Co., Tokyo, Japan) was used as a basal diet, which consists of 5.3% fat, 21.6% protein, 6.1% minerals, 2.9% fiber and 62.1% carbohydrate and others (
3.6 kcal/g). The major fatty acids present in MF were linoleic acid, oleic acid and palmitic acid. Administration of MF to each mouse was controlled (3 g/mouse/day) whereas water was available ad libitum during the experiment. AOM was obtained from Sigma (St Louis, MO).
Experimental procedures
Starting at 4 weeks of age, 10 homozygous db/db mice, 13 heterozygous db/+ mice, and 12 littermate controls (+/+) were given s.c. injections of AOM (15 mg/kg body wt) once weekly for 5 weeks to induce dysplastic and early neoplastic lesions of the colon. The remaining five animals of each genotype were injected with 0.2 ml of saline without AOM and served as controls. The food intakes of all the mice were restricted equally by feeding the same amount of MF during the experiment. All the mice were carefully observed daily under the food-restricted conditions. The experiment was terminated 10 weeks after the first injection of AOM (13 weeks of age) and all animals were killed. At autopsy, colons of all the mice were removed, cut open longitudinally and fixed in 10% buffered formalin. After removing the rectal sides (1 cm from the anus), the colons were cut into two portions (distal and proximal) and the distal colons were used in this study. They were embedded in paraffin blocks using an en face preparation technique (20) and processed for histopathological examination with hematoxylin and eosin (H&E) staining. Dysplastic and early neoplastic lesions in the colonic mucosa were identified microscopically according to the criteria described by Chang (21) and Risio et al. (22). Although we did not evaluate the formation of aberrant crypt foci (ACF) in the unsectioned colons, dysplastic lesions may include dysplastic ACF, but not hyperplastic ones. Epididymal fat tissues were also removed and weighed.
Leptin and insulin in blood
At killing, blood samples of saline-treated mice were collected for determination of glucose, leptin and insulin concentrations by enzyme immunoassay (EIA) according to the manufacturer's protocol (R&D systems, Minneapolis, MN).
Immunohistochemical analysis
Immunohistochemistry was performed using stain system kits (Dako, Kyoto, Japan; Zymed, South San Francisco, CA). Rabbit polyclonal antibodies against Ob-R (Santa Cruz Biotechnology, Santa Cruz, CA) and insulin-like growth factor-I receptor (IGF-IR) (Santa Cruz Biotechnology) were applied to the sections according to the manufacturer's protocols. For evaluation of the immunoreactivity cells were considered positive when definite cytoplasmic staining was identified.
Statistical analysis
Data were compared by ANOVA and post hoc tests. The results were considered statistically significant if the P values were <0.05.
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Results
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General observations
Food intake of the animals was carefully monitored every day and the total amount of food intake per animal was thought to be the same. The average body weights at the termination of the experiment (Figure 1) were respectively 20.7 ± 4.6 g in AOM-exposed db/db mice, 21.7 ± 2.0 g in AOM-exposed db/+ mice, 19.4 ± 1.6 g in AOM-exposed +/+ mice, 24.3 ± 0.8 g in saline-treated db/db mice, 22.9 ± 1.3 g in saline-treated db/+ mice and 22.6 ± 0.7 g in saline-treated +/+ mice (means ± SD). The body weights of saline-treated +/+ mice were lower than those of saline-treated db/db mice (P < 0.05) and there was a downward trend in the body weights of AOM-treated mice when compared with the corresponding saline-treated ones. In general, however, the body weights of all mice were controlled favorably. All animals tolerated the AOM injections except three of the db/db mice, four of the db/+ mice and two of the +/+ mice, which died during or soon after the period of AOM exposure, possibly because of a decreased maximum tolerated dose due to the AOM treatment.
Concentrations of serum leptin and other factors
Concentrations of leptin and insulin in the blood of db/db, db/+ and +/+ mice were measured by EIA. As shown in Table I, the levels of both leptin and insulin in db/db mice were significantly higher than those in db/+ or +/+ mice. In contrast, the levels of blood glucose were comparable among these mice. The results indicate that despite the food restriction, hyperleptinemia and hyperinsulinemia occurred in db/db mice, but not in db/+ mice. In support of the results, the epididymal fat weights in db/db mice were significantly higher when compared to db/+ and +/+ mice (Table I) and mRNA transcripts of the leptin gene in the fat tissues were up-regulated specifically in db/db mice (data not shown).
Dysplastic and early neoplastic lesions
At killing there were no macroscopic tumors in any colon. On the histological sections with H&E staining a number of dysplastic (Figure 2C) and early neoplastic (Figure 2E) lesions were detected under microscopic examination. As summarized in Table II, AOM treatment induced 29.3 ± 14.1 (mean ± SD) total lesions (dysplasia and early neoplasia) per cm2 of colon in db/db mice (Figure 2A), 13.8 ± 3.6 in db/+ mice and 15.9 ± 9.5 in +/+ mice (Figure 2B). There were 2.1- and 1.8-fold increases in the multiplicity of the total lesions in db/db mice when compared to db/+ and +/+ mice, respectively (P < 0.03 and P < 0.04). In saline-treated mice there were no microscopic lesions in the colonic mucosa. The results indicate that development of AOM-induced premalignant lesions of the colon was enhanced specifically in db/db mice.

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Fig. 2. Histology and immunohistochemistry of Ob-R and IGF-IR in AOM-exposed colonic mucosa. (A) Histology of the colonic mucosa in AOM-exposed db/db mice (x20). (B) Histology of the colonic mucosa in AOM-exposed db/+ mice (x20). Arrowheads (A and B) indicate dysplastic lesions and the arrow (A) points to an early neoplastic lesion. (C) A representative photograph of dysplastic lesions (x200). (D) Up-regulation of Ob-R expression in dysplasia. (E) A representative early neoplastic lesion (x100). (F) IGF-IR expression is up-regulated in early neoplasia. (A)(C) and (E), H&E staining; (D) and (F), immunohistochemistry of Ob-R and IGF-IR, respectively.
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Immunohistochemical analysis of Ob-R and IGF-IR
Our biochemical analysis showed that hyperleptinemia and hyperinsulinemia occurred in db/db mice. To determine the expression levels of the receptors for those hormones, immunohistochemistry using antibodies against Ob-R and IGF-IR was performed. Immunohistochemical expression of both Ob-R and IGF-IR was up-regulated in the cytoplasm of the dysplastic and neoplastic tissues (Figure 2D and F). The results suggest that up-regulation of the expression of these receptors might play a role in colon carcinogenesis and that hyperleptinemia and hyperinsulinemia could affect receptor-mediated signaling in db/db mice.
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Discussion
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In spite of the recent availability of several animal models for obesity, experimental evidence from these model systems suggesting an interactive mechanism of how obesity promotes colon carcinogenesis is relatively limited. In most of these animal models obesity is induced by dietary factors, such as a high fat diet or hyperphagia. Since the contribution of diet to cancer causation is generally thought to be high (23,24), it is possible that the dominant and complex features of the dietary factors may make it difficult to determine what factor is responsible for the relation between obesity and colon cancer. In this study we moderately restricted the food intake of all animals to equalize the involvement of dietary factors. As a result, the body weights of all the animals examined were controlled favorably, whereas leptin in the blood remained high specifically in db/db mice. This enabled us to assess the contribution of possible interactive factors such as hyperleptinemia to colon carcinogenesis. Although it does not suitably reflect human obesity, our model using db mice with food restriction is considered to be a novel tool for investigation of colon carcinogenesis in the settings of obesity and diabetes.
Our data suggest that development of AOM-induced premalignant lesions of the colon in db/db mice with hyperleptinemia was enhanced even under food-restricted conditions. The effect was actually expected and concordant with previous findings that AOM-induced colon carcinogenesis is enhanced in another obese model using Zucker rats (25,26). Concerning the mechanism(s), it is plausible that hyperleptinemia may be critical for this enhancement, because leptin acts as a growth factor in the colon (16) and appears to play a role in tumorigenesis of other organs, such as mammary gland (27). Leptin exerts its effects through the Ob-R, which has at least five splicing variants (Ob-Ra, Ob-Rb, Ob-Rc, Ob-Rd and Ob-Re) (12). In db/db mice a single point mutation in the C-terminal region of the Ob-R gene leads to a splicing abnormality of Ob-Rb (12). Consequently, Ob-Rb, which plays the main role in satiety signaling in the hypothalamus, is specifically deficient in these mice. On the other hand, the other splicing variants, Ob-Ra, Ob-Rc, Ob-Rd and Ob-Re, are preserved in db/db mice (12), although the function(s) of these receptors remains undetermined. Interestingly, a recent report showed that Ob-Ra, which is ubiquitously expressed in the periphery, performs signaling transduction (28). Furthermore, mitogen-activated protein kinase (MAPK), a protein kinase which plays a central role in regulating the activity of many nuclear transcriptional factors involved in inflammatory, immune and proliferative responses, has been shown to be one of the signal cascades of leptin mediated through the splicing variants of Ob-R (29). In addition, our immunohistochemical results showed that expression of Ob-R was up-regulated in AOM-induced lesions, suggesting a role in colon carcinogenesis. Taken together, there is a possibility that the enhancement of development of AOM-induced premalignant lesions of the colon in db/db mice may be caused by leptin-mediated signaling, such as through MAPK.
This study also showed that insulin in the blood was high specifically in food-restricted db/db mice. This result is consistent with previous findings that the expression of insulin is negatively regulated by the leptin pathway (30,31). Unexpectedly, the levels of blood glucose were comparable among the mice, suggesting that resistance to insulin might occur in db/db mice under these experimental conditions (32). There is accumulating evidence suggesting that hyperinsulinemia is involved in colon carcinogenesis as well as obesity and diabetes. Several epidemiological studies indicate that diabetic patients with hyperinsulinemia have increased risk for colon cancer (33). Additionally, a previous model assay showed that continuous injections of insulin promote AOM-induced colon carcinogenesis in rats (34). Hence, it seems likely that hyperinsulinemia in db/db mice enhanced the development of AOM-induced lesions in the present study. Regarding the mode of action, the current consensus assumes that the insulin-like growth factor-I (IGF-I) pathway plays a role in insulin-related tumor promotion in the colon (35). IGF-I binds to the IGF-IR, activates a signal cascade and triggers cell proliferation in several organs, including colon (36). Insulin at supra-physiological levels also binds to and activates the IGF-IR because of its homology with the insulin receptor (37,38). Furthermore, hyperinsulinemia was shown to indirectly increase bioavailability of IGF-I by regulating levels of IGF-binding proteins (36,39). In addition, our immunohistochemical data showed that IGF-IR expression was up-regulated in AOM-induced dysplastic and early neoplastic tissues of colon. This result was supported by a previous report showing that the IGF-IR is overexpressed in colon cancer in humans (40). Accordingly, it is possible that hyperinsulinemia in db/db mice activates the signaling cascades involving the IGF-IR, resulting in a proliferative response.
In conclusion, our data indicate that development of AOM-induced premalignant lesions of the colon was enhanced in db/db mice with hyperleptinemia and hyperinsulinemia. However, further studies will be necessary to reveal the specific determinants responsible for the correlation between obesity (and diabetes) and colon carcinogenesis.
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Acknowledgments
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The authors thank Kyoko Takahashi, Satomi Yasuda, Tomoko Kajita and Sonoko Nakatani for their excellent technical assistance and Yoshitaka Kinjo for animal care. This work was supported in part by Grants-in-Aid from the Ministry of Health, Labor and Welfare and Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan.
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Received July 23, 2003;
revised December 9, 2003;
accepted December 12, 2003.