Effects of calcium and vitamin D on MLH1 and MSH2 expression in rectal mucosa of sporadic colorectal adenoma patients

Abstract

To further clarify and develop calcium and vitamin D as chemopreventive agents against colorectal cancer in humans and develop modifiable biomarkers of risk for colorectal cancer, we conducted a pilot, randomized, double-blind, placebo-controlled, 2 x 2 factorial clinical trial to test the effects of calcium and vitamin D(3), alone and in combination, on key DNA mismatch repair proteins in the normal colorectal mucosa. Ninety-two men and women with at least one pathology-confirmed colorectal adenoma were treated with 2.0 g/d calcium or 800 IU/d vitamin D(3), alone or in combination, versus placebo over 6 months. Colorectal crypt overall expression and distribution of MSH2 and MLH1 proteins in biopsies of normal-appearing rectal mucosa were detected by automated immunohistochemistry and quantified by image analysis. After 6 months of treatment, MSH2 expression along the full lengths of crypts increased by 61% (P = 0.11) and 30% (P = 0.36) in the vitamin D and calcium groups, respectively, relative to the placebo group. The estimated calcium and vitamin D treatment effects were more pronounced in the upper 40% of crypts (differentiation zone) in which MSH2 expression increased by 169% (P = 0.04) and 107% (P = 0.13) in the vitamin D and calcium groups, respectively. These findings suggest that higher calcium and vitamin D intakes may result in increased DNA MMR system activity in the normal colorectal mucosa of sporadic adenoma patients and that the strongest effects may be vitamin D related and in the differentiation zone of the colorectal crypt.

Figures

Figure 1

Quantitative image analysis of MSH2 labeling optical density consists of several steps: a) finding eligible crypts (see text for details); b) manually tracing one side of the crypt (“hemicrypt”); c) automated division of the outline into segments of width of an average colonocyte; d) automated background-corrected densitometry of overall and segment-specific labeling of the biomarker and entering the results into the database.

Figure 2

Expression of MSH2 protein at standardized positions within the crypts of normal-appearing rectal mucosa in four treatment groups.* The Calcium, Vitamin D vs. Markers of Adenomatous Polyps Trial *Data points represent average within treatment group (by visit) batch-adjusted optical density at a particular standardized position in the crypt, and the curves are Loess smoothing curves (smoothing parameter 0.5).

Figure 3

Expression of MLH1 protein at standardized positions within the crypts of normal-appearing rectal mucosa in four treatment groups.* The Calcium, Vitamin D and Markers of Adenomatous Polyps Trial * Data points represent average within treatment group (by visit) batch-adjusted optical density at a particular standardized position in the crypt, and the curves are Loess smoothing curves (smoothing parameter 0.5).