There is strong evidence that the risk of colon cancer (CC) in humans is reduced by appropriate levels of physical exercise (27). Similar evidence has also been observed in experimental models of CC in rodents submitted to specific types of exercise (3,26). Recently, it has been observed that the inhibitory effects of exercise against CC are likely to occur from the early stages of the carcinogenic process, because low-intensity running training inhibits the development of aberrant crypt foci in rats treated with the carcinogen dimethyl-hydrazine (DMH) (8). Nevertheless, the mechanisms involved in the protective effect of exercise against cancer development remain largely unknown (2,7). This is an important issue because a better understanding of the mechanisms through which physical activity protects against CC would provide the basis for public health advice and for the development of novel intervention strategies (2). A recent systematic review of the literature identified that the main proposed mechanisms include changes in gastrointestinal transit time, altered immune function and prostaglandin levels, and changes in bile acid secretion, serum cholesterol, and gastrointestinal and pancreatic hormone profiles (24). The authors of this review conclude that there are currently few empirical data to support any of the hypothesized biological mechanisms for the protective effect of exercise on CC. They also consider that a greater understanding of the biological mechanisms involved will be an important step in developing exercise prescriptions targeted to reduce the burden of CC across different populations (24).
Inflammation is emerging as a unifying link between a range of exposures, including increasing age and greater body fatness, and neoplastic risk (2). The plasma levels of C-reactive protein, an inflammation marker, are associated with a subsequent risk of CC, and it is thought that inflammation may be involved at the early stage of colon tumor growth (19). Expression of inflammation-associated genes including cyclooxigenase-2 (COX-2) is upregulated in both inflamed mucosa and in colonic tumors (11). Furthermore, frequent and prolonged consumption of nonsteroidal antiinflammatory drugs (NSAIDs) seems to prevent formation or progression of CC in humans (16) and experimental animals (18). Interestingly, it has been hypothesized, but not confirmed, that appropriate levels of exercise may play an antiinflammatory role in the organism (4), and physical activity, especially when accompanied by lower-body fatness, may contribute to lower-bowel cancer risk by reducing the inflammatory stimulus experienced by the gut mucosa (3).
The carcinogen DMH induces an increase in the colonic epithelial cell proliferation in rats (20,30). Hyperproliferation is considered an early maker of CC risk (30). The carcinogen DMH also induces an increase in COX-2 expression in the colonic mucosa from very early stages of the colonic carcinogenesis (1), and we have recently observed that this increase in COX-2 expression occurs mainly in the colonic pericryptal cells (10). Thus, our aims in the present study were to confirm the existence of an early inhibitory effect of exercise against colon carcinogenesis by studying epithelial cell proliferation, and to verify the influence of the exercise on the expression of COX-2 in rats treated with DMH.
MATERIALS AND METHODS
Thirty-two male Wistar rats supplied by Ribeirão Preto School of Medicine, 30 d after birth, were housed one or two per cage in a temperature-controlled room at 24°C on average, and they were maintained on a 12:12-h light:dark cycle. The rats were provided with the same diet, a commercial chow purchased from Ribeirão Preto School of Medicine, and tap water, on an ad libitum basis. The animals were maintained in agreement with the guidelines of Committee on Care and Uses of Laboratory Animals of the National Research Council of the National Institutes of Health. Each rat's body weight was evaluated at the beginning and end of the experiment.
The animals were randomly divided into four groups, with eight animals in each one. Groups G1 and G3 were sedentary (controls), and groups G2 and G4 were submitted to exercise, performed as 8 wk of swimming training, 5 d·wk−1, according to the protocol of Venditti and Di Meo (28). Swimming was performed in a plastic container 100 cm high, filled to a depth of 60 cm with water, maintained at a temperature between 35 and 36°C. During the exercise protocols, the control group animals were kept in a small chamber containing about 3 cm of water maintained at 35°C (28). The groups G3 and G4 were given subcutaneous injections of DMH (50 mg·kg−1 body weight; Wako Pure Chemical Industries, Osaka, Japan) (19) immediately after been submitted to the exercise protocols (28). Fifteen days after neoplasic induction, the rats were sacrificed (5). At the autopsy, the large bowel was longitudinally opened as close as possible to the mesenteric border throughout its full extension. The distal colon and rectum were fixed in formalin, embedded in paraffin, and sampled for histological examination (hematoxylin and eosin staining).
Colonic mucosal sections were examined, using 3- to 5-μm-thick serial sections. For each case, 20-40 serial sections of crypts were used to investigate whole crypts from the mucosal surface to the crypt bottom. For immunohistochemical analysis, the labeled streptavidin biotin method was performed, using primary antibodies against PCNA and COX-2, as described previously (10,30). The primary antibodies were obtained from Novocastra Laboratories (Newcastle, UK).
Immunohistochemical staining was performed using an established avidin-biotin detection method by Novostain Universal Detection Kit (Novocastra Laboratories, Newcastle, UK) with some modifications. Four-micrometer-thick sections of paraffin-embedded tissue blocks were cut, mounted on positively charged glass slides, and dried in an oven at 56°C for 30 min. The sections were deparaffinized in xylene and then rehydrated in graded ethanol and water. Endogenous peroxidase was blocked by incubation in 3% hydrogen peroxide for 20 min. For all the immunohistochemical studies, the antigen retrieval was accomplished by pretreating the sections with a citrate buffer at pH 6.0 for 40 min at 98°C in a microwave oven and then allowing the sections to cool for 30 min at room temperature. Nonspecific endogenous protein binding was blocked using the prediluted serum. This step was followed by incubation with a biotin-labeled primary antibody solution in a damp chamber for two. The sections were then submitted to the following sequential treatments: staining with streptavidin/peroxidase complex reagent for 30 min, reaction with 3,3-diaminobenzidine tetrahydrochloride (Sigma-Aldrich Inc.), and counterstaining with hematoxylin followed by cover slipping. Sections of the tonsil served as positive controls. For negative controls, the primary antibody was omitted
To estimate colonic cell proliferation, in each colon the epithelial cell nuclei were counted in 100 colonic crypts. The PCNA-labeling index (PCNA-Li) was expressed as a ratio of positively stained nuclei to total nuclei counted per 100 crypts. A statistical analysis was performed using the ANOVA test. To estimate COX-2 expression, we have observed 100 colonic crypts and their surrounding connective tissue (10). The positive cells were scored, and the results were expressed as the number of COX-2-positive cells per colonic crypt (iCOX-2). Analysis was performed by two independent observers blind to the experimental group. No significant intraobserver bias was found.
Statistical analysis was performed by ANOVA test, followed by a post hoc Tukey's honestly significant difference test. A probability of P < 0.05 was considered statistically significant. The results were expressed as means ± SEM.
All the animals survived the experimental period and remained in good health. At the end of the experiment, the average body weight of the animals did not statistically differ between the two experimental groups submitted to exercise (G2 and G4) or between the two sedentary groups (G1 and G3). The body weight of groups G2 and G4 was significantly reduced when compared with the sedentary group G1 (7.1 and 7.3%, respectively). No macroscopic changes were observed in the colons.
The results of PCNA-LI are shown in Figure 1. Training group G3 presented a small, nonsignificant reduction of the PCNA-Li of the epithelium of the intestinal mucosa when compared with the sedentary G1 group. There was an increase in the PCNA-Li in both DMH-treated groups of rats. However, this increase was significantly attenuated in training group G4 (P < 0.01). Similar results were observed in relation to COX-2 expression, as shown in Figure 2.
In our experimental model, exercise was associated with an attenuation of the hyperproliferative response to the DMH. Because the epithelial hyperproliferation is regarded as a premalignant lesion, our results lend support to the proposed assumption that physical exercise may reduce the risk for development of CC. This finding is in accordance with a recent observation that a 12-month, moderate- to vigorous-intensity aerobic exercise intervention resulted in significant decreases in colon crypt cell proliferation indices in men (17).
We have also observed that exercise reduced the colonic expression of the COX-2 enzyme. It is known that colonic carcinogenesis is associated with increased COX-2 expression, which leads to excessive production of prostaglandin E2 (PGE2), which, in turn, forms a stimulatory loop with many biologic functions, including increasing cell proliferation (29). From our results, it is possible to hypothesize that the antiproliferation effect of exercise may be at least partially attributable to the inhibition of COX-2 expression.
Higher levels of colonic mucosal PGE2 have been observed in rodents with CC (9) and in individuals with colorectal polyps or cancer (22). This can be avoided by the use of NSAIDs (16). Furthermore, higher levels of physical activity have been associated with significant reductions in rectal mucosal PGE2 levels, suggesting that the mechanisms both for NSAIDs and for physical activity underlie similar activities-that is, COX-1 and COX-2 inhibition (15). Our findings strongly support this hypothesis.
The mechanisms involved in the exercise-induced reduction of COX-2 expression are unknown and warrant further investigation. The gut is very sensitive to exercise. During exercise, blood will primarily be shunted to the skin and exercising muscles, at the expense of the gastrointestinal tract. Thus, an exercise-induced inflammatory response arises primarily as a result of local tissue ischemia (23). On the other hand, this inflammatory stimulus may trigger long-term antiinflammatory mechanisms, which are partly mediated by muscle-derived IL-6. Physiological concentrations of IL-6 stimulate the appearance in the circulation of the antiinflammatory cytokines IL-1ra and IL-10 and inhibit the production of the proinflammatory cytokine TNF. This mechanism has been proposed to explain how the antiinflammatory effects of exercise may offer protection against TNF-induced insulin resistance (21). We propose that a similar mechanism could also protect against chemical carcinogen-associated inflammation. This hypothesis is supported by the concept that exercise leads to a balanced equilibrium between inflammatory and antiinflammatory responses (6). Furthermore, a similar mechanism has been observed in relation to the oxidative stress elicited by exercise. Contraction-induced production of reactive oxygen species has been shown to cause oxidative stress to skeletal muscle. As an adaptive response, muscle antioxidant defense systems are upregulated after heavy exercise. Nuclear factor kappaB (NFKB) is a major oxidative stress-sensitive signal transduction pathway in mammalian tissues. Activation of the NFKB signaling cascade has been shown to enhance the gene expression of important enzymes, such as mitochondrial superoxide dismutase, to maintain cellular oxidant-antioxidant homeostasis during exercise (13,14). Thus, it was proposed that "a major benefit of nonexhaustive exercise is to induce a mild oxidative stress that stimulates the expression of certain antioxidant enzymes…" (12). Interestingly, it has recently been observed that regular exercise adaptive responses include attenuation of an increased NF-kappaB activation (25). It is likely that this could explain the attenuation of the increase in COX-2 expression that we found, because COX-2 expression is stimulated by NF-kappaB.
From our findings, we conclude that exercise training attenuates the DMH-related increase in epithelial cell proliferation and mucosal COX-2 expression in the rat colon, suggesting that this may be an important mechanism of the complex relationship between exercise and CC.
Part of this work was supported by CAPES, CNPq, and FAPESP (grant 2001/14227-0). The authors would like to thank Mrs. R.O. Lopes, Mrs. F. Martinello, and Mrs. A. Crescenzi for technical support, and Mrs. Roselyne Lesur for the correction to the English language.
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