Nutr Res Pract.  2017 Aug;11(4):281-289. 10.4162/nrp.2017.11.4.281.

Increase in dietary protein content exacerbates colonic inflammation and tumorigenesis in azoxymethane-induced mouse colon carcinogenesis

• Tak KH
• Ahn E
• Kim E

• KMID: 2385466
• DOI: http://doi.org/10.4162/nrp.2017.11.4.281

Abstract

BACKGROUND
/OBJECTIVE: The incidence of colorectal cancer (CRC) has been attributed to higher intake of fat and protein. However, reports on the relationship between protein intake and CRC are inconsistent, possibly due to the complexity of diet composition. In this study, we addressed a question whether alteration of protein intake is independently associated with colonic inflammation and colon carcinogenesis.
MATERIALS/METHODS
Balb/c mice were randomly divided into 4 experimental groups: 20% protein (control, 20P, 20% casein/kg diet), 10% protein (10P, 10% casein/kg diet), 30% protein (30P, 30% casein/kg diet), and 50% protein (50P, 50% casein/kg diet) diet groups and were subjected to azoxymethane-dextran sodium sulfate induced colon carcinogenesis.
RESULTS
As the protein content of the diet increased, clinical signs of colitis including loss of body weight, rectal bleeding, change in stool consistency, and shortening of the colon were worsened. This was associated with a significant decrease in the survival rate of the mice, an increase in proinflammatory protein expression in the colon, and an increase in mucosal cell proliferation. Further, colon tumor multiplicity was dramatically increased in the 30P (318%) and 50P (438%) groups compared with the control (20P) group.
CONCLUSIONS
These results suggest that a high protein diet stimulates colon tumor formation by increasing colonic inflammation and proliferation.

Keyword

Diet; casein; colonic neoplasms; colitis

MeSH Terms

Animals
Body Weight
Carcinogenesis*
Caseins
Cell Proliferation
Colitis
Colon*
Colonic Neoplasms
Colorectal Neoplasms
Diet
Dietary Proteins*
Hemorrhage
Incidence
Inflammation*
Mice*
Sodium
Survival Rate
Caseins
Dietary Proteins
Sodium

Figure

• 

  Fig. 1 Schematic representation of the experiment. Twenty-nine 4-week-old female Balb/c mice were acclimated for 1 week and then randomly divided into four diet groups based on AIN-76A diet composition: control (20P; 20% casein, n = 5), 10P (10% casein, n = 8), 30P (30% casein, n = 8), and 50P (50% casein, n = 8). Carcinogenesis was initiated with a single intraperitoneal injection of 12.5 mg/kg body weight AOM and was promoted with 3 cycles of DSS in drinking water 1 week after AOM injection in the mice of all four groups. The animals were received 2% DSS in their drinking water for one cycle and were then switched to 1% DSS drinking water for the remaining two cycles because of the severity of the disease. Each cycle lasted five days and the cycles were separated by 16 days. Experimental diet was fed to the mice after AOM initiation and was continued for 11 weeks. AOM, azoxymethane; DSS, dextran sodium sulfate.

• 

  Fig. 2 Changes in body weight. Body weight was measured once a week during the experimental period. Time points of AOM injection and DSS supplementation are indicated by arrows. Values are presented as the mean ± SE. Means with different letters are significantly different at P < 0.05 by Duncan's multiple range test. NS, not significant; AOM, azoxymethane; DSS, dextran sodium sulfate; 20P, 20% casein; 10P, 10% casein; 30P, 30% casein; 50P, 50% casein contents of diet.

• 

  Fig. 3 Disease activity index and the survival rate of mice. (A) Clinical scores of mice in each experimental group were monitored every day. 0, no weight loss, no occult blood in the stools, and normal stool consistency; 1, weight loss of 1-5%, no occult blood, and normal stool consistency; 2, 5-10% weight loss, positive for fecal occult blood, and loose stools; 3, 10-20% weight loss, positive for fecal occult blood, and loose stools; 4, greater than 20% weight loss, gross rectal bleeding, and diarrhea. Means with different letters are significantly different at P < 0.05 by Duncan's multiple range test. (B) The survival rate of mice is expressed as a percentage of live mice in each group every week. 20P, 20% casein; 10P, 10% casein; 30P, 30% casein; 50P, 50% casein contents of diet.

• 

  Fig. 4 Weight and length of large intestine. Colons were obtained 10 days after the last DSS cycle and their weight (A) and length (B) were measured as described in materials and methods. Values are presented as the mean ± SE. Means with different letters are significantly different at P < 0.05 by Duncan's multiple range test. 20P, 20% casein; 10P, 10% casein; 30P, 30% casein; 50P, 50% casein contents of diet.

• 

  Fig. 5 Expression of inflammatory proteins. The large intestine from cecum to rectum was divided into 3 parts and the distal parts were removed and homogenized. The inflammatory proteins COX-2 (A) and iNOS (B) levels were determined by immunoblotting with the appropriate antibodies. Values are presented as the mean ± SE. Means with different letters are significantly different at P < 0.05 by Duncan's multiple range test. 20P, 20% casein; 10P, 10% casein; 30P, 30% casein; 50P, 50% casein contents of diet; COX-2, cyclooxygenase-2; iNOS, inducible nitric oxide synthase.

• 

  Fig. 6 Effect of HPD on intestinal epithelial hyperplasia and proliferation. The distal part of large intestine was removed and fixed in 10% formalin. (A) Tissue sections were stained with hematoxylin and eosin and photographed at 100X: (a) 20P (control), (b) 10P, (c) 30P, and (d) 50P groups. (B) Mucosal thickness was measured microscopically. (C) Tissue sections were immunostained for PCNA and photographed at 400X: (a) 20P (control), (b) 10P, (c) 30P, and (d) 50P groups. (D) The labeling index was calculated as the percentage of stained cells to the total number of cells in the colon epithelium. Means with different letters are significantly different at P < 0.05 by Duncan's multiple range test. PCNA: proliferating cell nuclear antigen. 20P, 20% casein; 10P, 10% casein; 30P, 30% casein; 50P, 50% casein contents of diet.

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