Prevention of preneoplastic lesions by dietary vitamin D in a mouse model of colorectal carcinogenesis

Abstract

Colorectal cancer (CRC) is one of the leading causes of cancer morbidity and mortality in Western countries. One of the risk factors for colorectal tumorigenesis is vitamin D insufficiency. The aim of this study was to establish whether increasing dietary vitamin D intake can prevent or delay development of chemically induced preneoplastic lesions in the colon of mice. We fed six weeks old female C57BL/6J mice (n=28) with increasing vitamin D3 concentrations (100, 400, 1000, 2500, 5000IU/kg diet). To induce dysplasia, a preneoplastic lesion, we injected mice with the carcinogen azoxymethane (10mg/kg) intraperitoneally, followed by three cycles of 2% dextran sodium sulfate salt, a tumor promoter, in the drinking water. To test our hypothesis that high vitamin D intake prevents formation of preneoplastic lesions, we have investigated the effect of increasing dietary vitamin D on development of premalignant colorectal lesions, serum 25-hydroxyvitamin D3 (25-D3) levels, and expression of renal vitamin D system genes. Dietary vitamin D concentration correlated inversely with dysplasia score (Spearman's correlation coefficient, ρ: -0.579, p=0.002) and positively with serum 25-D3 levels (ρ: 0.752, p=0.001). Increasing dietary vitamin D concentration beyond 1000IU/kg led to no further increase in circulating 25-D3 levels, while the dysplasia score leveled out at ≥2500IU/kg vitamin D. High dietary vitamin D intake led to increased renal mRNA expression of the vitamin D catabolizing enzyme cyp24a1 (ρ: 0.518, p=0.005) and decreased expression of the vitamin D activating enzyme cyp27b1 (ρ: -0.452, p=0.016), protecting the body from toxic serum levels of the active vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25-D3). Our data showed that increasing dietary vitamin D intake is able to prevent chemically induced preneoplastic lesions. The maximum impact was achieved when the mice consumed more than 2500IU vitamin D/kg diet. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Fig. 1

High dietary vitamin D decreases dysplasia score and increases serum 25-D₃ levels. (A) Effect of diets containing increasing amounts of vitamin D on dysplasia score. Score was calculated by adding percentage of low dysplasia region multiplied by 2 and percentage of high dysplasia regions multiplied by 3. Circles represent colonic dysplasia scores of individual mice, horizontal lines represent median. (B) Effect of vitamin D content of the diet on serum 25-D₃ levels. Horizontal lines represent median. Discontinous lines are the borders of vitamin D deficiency (<20 ng/ml), and sufficiency (≥30 ng/ml).

Fig. 2

High dietary vitamin D prevents preneoplastic changes in colon of mice. Hematoxylin–Eosin staining of (A) normal colon of a mouse fed with a diet containing 5000 IU/kg and (B) colon with high grade dysplasia from a mouse fed 100 IU vitamin D/kg diet.

Fig. 3

Ki67 protein expression is increased in dysplastic region compared with normal mucosa. Immunohistochemistry of a colon from a mouse fed with 100 IU/kg vitamin D. Proliferation marker Ki67 was highly expressed in dysplastic region (right panel) compared with the adjacent normal mucosa (left panel) of the same mouse.

Fig. 4

High dietary vitamin D increases cyp24a1, decreases cyp27b1 and does not affect vdr expression. Effect of increasing vitamin D concentration in the diet on renal cyp24a1 (A), cyp27b1 (B) and vdr (C) expression. Circles represent relative expression of individual mice, horizontal lines represent median.