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Aerobic Training Activates Interleukin 10 for Colon Anticarcinogenic Effects

FRAJACOMO, FERNANDO TADEU1; KANNEN, VINICIUS1; DEMINICE, RAFAEL2; GERALDINO, THAIS HERRERO3; PEREIRA-DA-SILVA, GABRIELA3; UYEMURA, SERGIO AKIRA4; JORDÃO-JR, ALCEU AFONSO2; GARCIA, SERGIO BRITTO1

Author Information
Medicine & Science in Sports & Exercise: September 2015 - Volume 47 - Issue 9 - p 1806-1813
doi: 10.1249/MSS.0000000000000623

Abstract

Colorectal cancer is the third most common malignancy and the cause of tumor-related deaths in the United States of America (47). Aerobic physical exercise has been shown to reduce the risk for colon cancer in humans and rodents (1,7,12,21,25,30). Resistance training has been observed reducing the risk of recurrence of colon cancer (10). The American Cancer Society Guidelines thus advise adults to do moderate aerobic exercises about 150 min·wk−1 or 75 min of vigorous exercises in addition to up to 3 d of resistance training (34). However, to prevent cancer, deeper mechanistic understanding on the relation between exercise and carcinogenesis will provide a more accurate based prescription for physical training.

Physical exercise markedly modulates oxidative status in different organs. Studies have shown that either pro- or antioxidant effects of physical exercise depend on its timing, intensity, and modality (43,50). For instance, enhancement of lipid peroxidation by physical exercise has been reported in humans (15,23) and high-intensity exercise has been associated with a marked increase in total antioxidant capacity in athletes (36). High-intensity training, however, decreased lipid peroxidation and total antioxidant capacity in master athletes that were competing for at least 21 yr (51).

Oxidative stress may be both a cause and consequence of inflammation, which is closely related to the development of tumors (26). We reported that a similar physical training reduces carcinogenesis and inflammatory markers in the colon (21) Nevertheless, the mechanisms by which exercise modulates colonic inflammation remain poorly known.

Indeed, regulatory effects of inflammatory cells on colon mucosa are largely dependent on the synthesis of cytokines by the immune system. Cytokines may be associated either with tumor promotion or tumor suppression in the colon tissue (39). For example, interleukin 6 (IL-6), IL-17, IL-12, and tumor necrosis factor (TNF)-α contribute to a procarcinogenic microenvironment, whereas transforming growing factor β and IL-10 suppress carcinogenesis (39). IL-10 is emerging as a key mechanism counteracting proinflammatory and protumoral stimulus in the colon. IL-10 negatively modulates not only inflammatory bowel diseases but also early and late colon carcinogenic events (5,27). IL-10 knockout mice (IL-10−/−) were reported to be developing chronic intestinal inflammation, colon preneoplastic lesions, and colorectal adenocarcinomas spontaneously (5,22,27). Having T and regulatory T cells as a main expressing source of IL-10, this cytokine was found to modulate immune cellular infiltration within colonic polyps and an intestinal microbiota promoting cancer (22). When IL-10 was replaced to its knockout mice, intestinal inflammation was abrogated and colon carcinogenesis development was reduced (5).

Animal models have enabled cancer research to uncover several new mechanisms in colon carcinogenesis. For instance, the carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) successfully recapitulates in rodents the development of colon carcinogenesis among humans (3,14). MNNG is a topic-acting carcinogen, which promotes the formation of preneoplastic lesions in colonic epithelia, such as dysplastic aberrant crypt foci (ACF) (31). The expansion of these preneoplastic lesions requires enhanced oxidative stress and inflammatory signaling (35,45). Therefore, we sought to investigate the effects of aerobic and resistance trainings on MNNG-induced dysplastic ACF formation in the colon of mice. Our results suggest that only aerobic training has anticarcinogenic effects in the colon and that such protective activity is related to the IL-10 activity.

MATERIALS AND METHODS

Animals

Sixty-two male BALB/c (25 ± 2 g; 7–8 wk), 32 male C57/BL6 (25 ± 2 g; 7–8 wk), and 28 male IL-10−/− breeding pairs, on C57/BL6 background, were purchased from Jackson Laboratories (stock number 002251), from which colonies were established at the University of São Paulo (25 ± 2 g; 7–8 wk). The IL-10−/− mouse model was engineered on 129P2/OlaHsd-derived E14-1 embryonic stem cells electroporated with a targeting vector (24-bp linker in a neoexpression cassette) for the IL-10 gene sequence (codons 5–55 of the exon 1 were replaced). C57BL/6 blastocysts received mutant embryonic stem cells. Colonies were then established breeding wild-type C57BL/6 females with chimeric male offspring. Back-crossing the mutant colony to C57BL/6 J mice for at least 10 generations reduced the 129P2 genetic background. Other 15 generations were produced before current experiments were performed. Mice were housed in controlled a environment within plastic cages (55% humidity, 12-h light/dark cycle, and 22°C ± 0.5°C). Mice were acclimated for 1 wk before starting the experiments. Then, mice were randomly divided into experimental groups. All experiments were performed according to the protocol approved by the Ethical Committee in Animal Care and Use at the Ribeirao Preto Medical School, University of São Paulo (#013/2011 and #070/2014).

Physical Exercise Programs

Aerobic training

Swimming is a common exercise modality in rodent disease models (11). In mice, swimming has been reported preventing secondary stressful sources from treadmill-related foot injury (49). Current protocol was based on Venditti and Di Meo’s (49) previous description. Thus, BALB/c, C57/BL6, and IL-10−/− mice swam in experimental pools (100 cm (height) × 60 cm (width) × 65 cm (length)) filled with warm water (32°C ± 2°C; height, 50 cm). Swimming period was progressively increased from 20 (first week) to 60 min (third week), a training period kept steady for the next 5 wk. Altogether the mice swam 5 d·wk−1 during 56 d (total, 40 sessions) (see Figure, Supplemental Digital Content 1A, Evolution of the aerobic training, https://links.lww.com/MSS/A486).

Resistance training

Protocol was based on previous reports (28,38). To climb a ladder (80° inclination; 1-cm space between steps, and 50-cm height) (see Figure, Supplemental Digital Content 1B, Mouse training ladder, https://links.lww.com/MSS/A486), each BALB/c mouse performed 10 ± 2 dynamic movements. During each training session, 7 ± 1 climbs were performed by each mouse (2 min for recovery was given between each climbing repetition). Training was repeated three times a week during 56 d (24 training sessions in total). However, a maximal training load had to be established for resistance training (50% of the previous load, 75%, 90%, 100%, and 100% + 3.0 g). Thus, mice were trained to voluntarily climb the ladder without load attached to their tails during the first three sessions (first week). In the first training at the second week, a maximal training load test was performed to determine the performance of each mouse. At this moment, each mouse had 50% of its body weight attached to the proximal portion of its tail before climbing (latex rubber band). During the next three climbs, training load was progressively increased to reach 100% body weight of each mouse (75%, 90%, and 100%). To each mouse climbing the ladder with 100% of its body weight, a 3-g training-load was added for the next 2 ± 2 climbs. The maximal training load test provided enough data to calculate the training load for each mouse (maximal training load vs body weight variations). Then, resistance training was performed during the next 6 wk (see Figure, Supplemental Digital Content 1C and 1D, Evolution of the resistance training, https://links.lww.com/MSS/A486).

Experimental Design and Sacrifice

Experiment 1

BALB/c mice were either exposed or not to the carcinogen MNNG (four successive dosages of MNNG (5 mg·mL−1; intrarectal deposits of 100 μL; Sigma-Aldrich) twice a week for 2 wk) according to our previous description (31). MNNG-unexposed groups were as follows: sedentary (S, n = 6), aerobic (A, n = 8), and resistance (R, n = 8) (see Figure, Supplemental Digital Content 1C, Training evolution for the resistance protocol in carcinogen-unexposed mice, https://links.lww.com/MSS/A486). MNNG-exposed mice (M) were divided into three other groups (sedentary group (SM, n = 8), aerobic group (AM, n = 8), and resistance group (RM, n = 8) (see Figure, Supplemental Digital Content 1D, Training evolution for the resistance protocol in carcinogen-exposed mice, https://links.lww.com/MSS/A486)).

Experiment 2

IL-10−/− mice, together with their respective wild-type background (C57/BL6 mice), were randomly divided into the following groups: C57/BL6 (S, n = 8; A, n = 8; M, n = 8; and AM, n = 8) and IL10−/− mice (S, n = 8; A, n = 8; M, n = 5; and AM, n = 7).

All training protocols were applied 14 d after the first MNNG exposure. All mice were sacrificed in a carbon dioxide chamber 70 d after the first carcinogen exposure (see Figure, Supplemental Digital Content 1E, Percentage of survival by the Kaplan–Meyer test, https://links.lww.com/MSS/A486). Cardiac puncture was used for blood collection (21-gauge × 1.5″ needle; 1-mL syringe Ultra-fineTM II; Becton, Dickinson, and Company). Blood was sampled in tubes and centrifuged at 1106.82g for 10 min. Colon was collected from individual autopsies, in which samples were divided into snap-frozen (liquid nitrogen, −196°C) and fixed paraformaldehyde (4%) samples. The right hind legs had their skin removed; then, their soleus and gastrocnemius muscles were dissected, weighed, and snap-frozen. All samples were stored at −80°C until analysis.

Biochemical Analysis

Full descriptions for all following analyses are given in the Supplemental Digital Content. Thus, creatine kinase (CK; serum (U·L−1)) (see Document, Supplemental Digital Content 2, CK analysis, https://links.lww.com/MSS/A487), thiobarbituric acid reactive substances (TBARS; serum, μM·L−1) (see Document, Supplemental Digital Content 3, TBARS analysis, https://links.lww.com/MSS/A488), and total glutathione levels (GSH; serum, mM·L−1) (see Document, Supplemental Digital Content 4, GSH analysis, https://links.lww.com/MSS/A489) were biochemically analyzed.

Histopathological Analysis

Colon samples were stained with hematoxylin and eosin according to our previous description (31). To avoid observer bias, all slides were coded before microscopic analysis (Leica DM 2500; Leica Mikrosysteme Vertrieb, Germany; equipped with Leica DC 300 FX camera). Dysplastic lesions were identified at 20× magnification (pathological features ranging from mild to severe dysplasia). Confirmation of the dysplastic features was carried at 40× magnification for all samples. Colon morphometry was performed with Leica Application Suite (LAS) version 3.7 software, which determined colonic area automatically. Relative values for total dysplastic lesions were calculated as their total number per colonic sample areas (cm2 or mm2).

Immunohistochemistry

Antibody staining was performed according to our previous description (31,33). Anti-proliferating cell nuclear antigen (PCNA; clone PC 10 at 1:100) and anti-cyclooxygenase 2 (COX-2; clone 4H12 at 1:200) antibodies were purchased from Novocastra®. Positive reactions were displayed with Picture-MAX Polymer Kit (Invitrogen). Positive reactions for PCNA showed a brown precipitate at the nucleus, whereas COX-2 positivity was found at the cytoplasm. Proliferation was determined as the ratio between stained and unstained cells in colonic crypts. Enumerating the subepithelial COX-2–positive cells was shown as their number per counted intercrypt space.

Enzyme-Linked Immunosorbent Assay

Frozen colonic samples (100 mg) were homogenized mechanically (homogenization buffer: 1 M·L−1 of Tris–HCl, 3 M·L−1 of NaCl, and 10% Triton (supplemented with protease cocktail (Sigma-Aldrich)). Samples were centrifuged at 18.300g for 30 min at 4°C. Supernatants were frozen at −80°C until assay. According to the manufacturers’ guideline, IL-12, IL-10, TNF-α, and interferon gamma were quantified using enzyme-linked immunosorbent assay OptEIA kit (Biosciences Pharmingen).

Statistical Analysis

Data were analyzed using a GraphPad Prism 5 (Graph Pad Software Inc., San Diego, CA). Two-way ANOVA test (Bonferroni post hoc test) was used to separately analyze carcinogen-exposed and -unexposed mice. One-way ANOVA test (Kruskal–Wallis post hoc test) was applied to compare dysplastic lesions in carcinogen-exposed groups. The Mann–Whitney test was applied for comparisons between two groups only. The Kaplan–Meyer test was applied to calculate survival. P < 0.05 was considered to be statistically significant. All values are expressed as mean ± SD.

RESULTS

General effects of aerobic and resistance trainings

We previously reported that physical exercise induces protective effects in the colon of carcinogen-exposed rats (21). Here, we investigated the effects of aerobic and resistance trainings on the development of colon preneoplastic lesions for 8 wk (see Figure, Supplemental Digital Content 1A–D, Representation of exercise trainings, https://links.lww.com/MSS/A486). No significant mortality was observed throughout our experiments (see Figure, Supplemental Digital Content 1E, Percentage of survival, https://links.lww.com/MSS/A486). Although mice showed no significant weight differences at the beginning of experiments, carcinogen-exposed mice were found to be significantly heavier than the unexposed group on the 10th experimental week (Table 1) (P = 0.004, Mann–Whitney test). Resistance training increased the body weight of the carcinogen-unexposed group significantly (P < 0.05, two-way ANOVA test), whereas aerobic training decreased among the exposed mice (Table 1) (P < 0.01, two-way ANOVA test). We did not observe any significant changes in relative weight for the heart, gastrocnemius, or soleus (Table 1).

T1-6
TABLE 1:
Tissue weight.

Carcinogen exposure significantly increased the serum CK levels (P = 0.01, Mann–Whitney test), whereas such effect was abrogated by both aerobic and resistance physical exercises (Table 2) (P < 0.05, two-way ANOVA test). Moreover, carcinogen exposure enhanced the formation of lipid hydroperoxide reactive species 48% (TBARS), which was significantly suppressed by both physical trainings (Table 2) (P < 0.01 and P < 0.001, two-way ANOVA test). These two types of physical trainings further upregulated total GSH levels in carcinogen-unexposed groups (P < 0.05 and P < 0.01), whereas only aerobic training significantly enhanced these antioxidant levels among the exposed mice (Table 2) (P < 0.05, two-way ANOVA test).

T2-6
TABLE 2:
Serum biochemical analyses.

Effects of aerobic and resistance trainings on colon preneoplastic events

Aerobic training reduced the number of colon dysplastic lesions to 36% of the carcinogen-exposed sedentary group (P < 0.05, one-way ANOVA test). Such protective effect was not observed among resistance-trained mice (Fig. 1A and B). Moreover, aerobic-trained mice showed decreased subepithelial COX-2 expression to 20% of those of the carcinogen-exposed sedentary group (P < 0.05, two-way ANOVA test) (Fig. 1C) (see Figure, Supplemental Digital Content 5A–C, Representative pictures for subepithelial COX-2 expression, https://links.lww.com/MSS/A490). Interestingly, aerobic exercise training was related to a significant increase in the IL-10 levels among carcinogen-exposed mice (Fig. 1D–G) (P < 0.001, two-way ANOVA test).

F1-6
FIGURE 1:
Effects of physical training on the development of colon preneoplastic lesions. A, Representative pictures for normal colonic epithelia (A.1), single aberrant dysplastic crypt (AC) (A.2; green sectioned line indicates location), and an ACF with more than four AC (A.3). Pictures were taken at 40× magnification. Scale bars are inserted (25 μm). B, Relative values for preneoplastic lesions are shown per square centimeter (b P < 0.05 vs SM group; one-way ANOVA test and Kruskal–Wallis post hoc test). C, Relative values for COX-2 expression in colonic subepithelial areas (b P < 0.001 vs SM group; two-way ANOVA test and Bonferroni post hoc test). D, Interferon-gamma (**P < 0.001 (A) vs S group; and *P < 0.01 (R) vs S group; two-way ANOVA test and Bonferroni post hoc test). E, IL-12 (*P < 0.05 vs S group; two-way ANOVA test and Bonferroni post hoc test). F, TNF-α (P > 0.05). G, IL-10 (bb P < 0.001 vs SM group; two-way ANOVA test and Bonferroni post hoc test) levels determined by enzyme-linked immunosorbent assay. S (n = 6), A (n = 8), and R (n = 8). M represents MNNG-induced groups (SM, n = 8; AM, n = 6; and RM, n = 7). Data are shown as mean ± SD.

IL-10 is required for antipreneoplatic effects of aerobic training in the colon

Considering that aerobic training might protect from colon carcinogenesis enhancing IL-10 expression, we subjected carcinogen-exposed and -unexposed wild-type and IL10−/− mice to aerobic training. None of the carcinogen-unexposed mice showed any carcinogenic event in the colon. However, carcinogen-exposed IL-10−/− mice developed 20% more dysplastic lesions than its wild-type background (C57/BL6 (1.68 ± 0.91) vs IL-10−/− (8.69 ± 8.3 total dysplastic lesions per square millimeter)) (P = 0.01, Mann–Whitney test). Development of colon dysplastic lesions were again decreased in wild-type, carcinogen-exposed, aerobic-trained mice (Fig. 2A) (P = 0.0003, Mann–Whitney test). Antipreneoplastic effects of aerobic training were abrogated, knocking IL-10 reaction out from the colon tissue (Fig. 2B). Aerobic training further reduced the proliferation in wild-type carcinogen-exposed mice (Fig. 2C) (P < 0.05, two-way ANOVA test), but it did not protect IL-10−/− mice from that carcinogen-induced high proliferation in colonic crypts (Fig. 2D).

F2-6
FIGURE 2:
Antipreneoplastic effects of aerobic training are related to the colonic IL-10 activity. Relative values for preneoplastic lesions are shown per square millimeter in wild-type (A) (bb P = 0.0003 vs SM group; Mann–Whitney test) and IL-10−/− mice (B) (P > 0.05 vs SM group; Mann–Whitney test). Percentage of proliferating cells (PCNA) are shown per colonic crypt in wild-type (C) (b P <0.001 vs SM group; two-way ANOVA test and Bonferroni post hoc test) and IL-10−/− mice (D) (P > 0.05 vs SM group). Wild-type mice (S, n = 8; A, n = 8) (M represents MNNG-exposed groups (SM, n = 8; AM, n = 8). IL10−/− mice (S, n = 8; A, n = 8; M, n = 5; and AM, n = 7). Data are shown as mean ± SD.

DISCUSSION

Our findings support recent reports on the association of aerobic exercise with marked reduction of colon cancer risk in humans (7,8,13). Although studies differentiating the effects of aerobic (swimming) from resistance trainings (ladder climbing) on colon carcinogenesis are scarce, we found that resistance training did not act protectively against colon carcinogenesis as did the aerobic exercise. Notably, swimming and ladder climbing protocols were performed according to previous description and, therefore, any conclusions from the current findings should be bound strictly to these protocols (21,38,49).

We found that carcinogen exposure increased not only body weight but also muscle damage in mice. Indeed, the chemical carcinogen MNNG does not require metabolic activation to induce DNA damage and mutations (3). MNNG directly triggers DNA damage and induces early intense generation of reactive oxygen species (14), which promotes further DNA damage (41) as required for the full development of colon carcinogenesis (6,52). Here, we should remember that rectally administrated drugs might not undergo hepatic clearance through the first-pass metabolism. Despite the rectal upper portion drains into the portal system, about half of the rectal content is drained within the internal iliac vein from its lower part, bypassing hepatic metabolism through the inferior vena cava (18,19). Indeed, a great portion of the rectal content is delivered to the whole body. Moreover, carcinogen-induced muscle impairment is likely to have occurred in our experimental model. Different carcinogens promoting heart muscle damage in rats (29,48) and muscle damage being related to enhanced serum CK levels (17) were previously reported. Moreover, CK levels are enhanced in sedentary conditions (9). We believe that carcinogen exposure induced muscle damage, reducing spontaneous physical exercise, which potentially increased body weight in carcinogen-exposed physically untrained mice. Taking into account that CK analysis is an indirect biomarker for muscle damage, it limits any further hypothesis on how the muscle tissue was modulated in our experimental conditions.

Because we have observed that both aerobic and resistance trainings protected muscle tissue from carcinogen-induced damages, as shown by serum CK levels, it is unlikely that signaling derived from carcinogen-induced muscle damage may had a straightforward role in the anticarcinogenic effect of aerobic exercise in our experimental model. On the other hand, it is known that aerobic exercise induces IL-6 production by muscle fibers and IL-6 stimulates the appearance in the circulation of anti-inflammatory cytokines such as IL-10 (4,42). This is consistent with our current findings, i.e., a marked increase in the colonic IL-10 levels among carcinogen-treated, aerobically trained mice and abrogation of colon carcinogenesis protection in IL-10−/− mice exposed to similar exercise (Fig. 2A and B). Current experiments yet revealed that the resistance exercise was not related neither to anticarcinogenic effects nor IL-10 modulation in the colon. Recently, it has been reported that college-age men exhibited decreased IL-10 serum levels after 12 wk of resistance exercise training (32).

Organ-to-organ crosstalk involving muscle contraction at the molecular level is emerging as a field related to exercise. The muscle fiber-derived cytokines or peptides produced and secreted during skeletal muscle contraction have been classified as myokines, which includes IL-6 and IL-10 (42). Inflammation has been exhaustively associated with cancer development (24,39). IL-10 is a pivotal regulative element to keep the colon tissue in homeostasis, as reducing its inflammation (16,26). High-oxidative stress levels and inflammation were found in the intestines of IL-10−/− mice (40). In carcinogen-exposed IL-10−/− mice, high COX-2 expression was observed in subepithelial areas (46). Interestingly, mild-intensity aerobic training decreased inflammation in cachectic tumor-bearing rats, in which the IL-10 levels were increased (37).

Our collective data clearly indicate a relevant role of IL-10 for anticarcinogenic effects of aerobic exercise in the colon. However, we think that current results should be interpreted with caution. Although an aerobic exercise IL-10 signaling has been observed to be protective against colon carcinogenesis (2), it cannot be expected as a rule. For instance, exhaustive exercise was reported to reduce muscular inflammation, whereas it induced proinflammatory effects on adipocytes (44). Moreover, a similar physical exercise modality (aerobic training) was previously shown to have either pro- or antipreneoplastic effects on the colon, depending on its intensity (20,21). On the other hand, resistance exercise has been consistently reported to present beneficial effects in patients with cancer (53) and eventual protective effects of resistance exercise against carcinogenesis in different experimental conditions from the current study may not be ruled out.

Taken together, we suggest that regular aerobic training (swimming), but not resistance training (ladder climbing), promotes antipreneoplastic effects in the colon by enhancing IL-10 activity. In either physiological or carcinogenic conditions, new studies should distinguish swimming effects from those of the ladder climbing on muscle and colon tissues. Further efforts should nevertheless be applied to unveil the role played by the muscular system and eventual novel molecular mechanisms modulated by each of these physical trainings against colon carcinogenesis.

The authors would like to thank Mrs. R. O. Lopes, Ms. P. B. Ovídio, and Mr. J. P. Souza for their excellent technical assistance and Ms. Rosylene Lesur for correction of the English language.

Financial support was provided by the Coordination for the Improvement of Higher Education Personnel (CAPES), National Council for Scientific and Technological Development (CNPQ), and University of São Paulo, Brazil. None of funding agencies had any role in this study.

No conflict of interest, financial or otherwise, is declared by the authors.

The results of the present study do not constitute endorsement by the American College of Sports Medicine.

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Keywords:

COLON CANCER; PRENEOPLASTIC LESIONS; INFLAMMATION; PHYSICAL TRAINING

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