Effects of vitamin D and calcium supplementation on markers of apoptosis in normal colon mucosa: a randomized, double-blind, placebo-controlled clinical trial

Abstract

To further clarify and/or develop calcium and vitamin D as chemopreventive agents against colorectal cancer in humans, understand the mechanisms by which these agents reduce risk for the disease, and develop "treatable" 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 D3, alone and in combination on markers of apoptosis, 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 D3, alone or in combination, versus placebo over 6 months. Overall expression and colorectal crypt distributions of Bcl-2 (an apoptosis inhibitor) and Bax (an apoptosis promoter) in biopsies of normal-appearing rectal mucosa were detected by automated immunohistochemistry and quantified by image analysis. After 6 months of treatment, Bax expression along the full lengths of crypts increased 56% (P = 0.02) in the vitamin D group and 33% in both the calcium (P = 0.31) and calcium plus vitamin D (P = 0.36) groups relative to the placebo group. The vitamin D treatment effect was more pronounced in the upper 40%, or differentiation zone, of crypts (80%; P = 0.01). There were no statistically significant treatment effects on Bcl-2 expression. Overall, these preliminary results suggest that calcium and vitamin D, individually or together, may enhance apoptosis in the normal human colorectal epithelium, and the strongest treatment effects may be vitamin D related and in the upper sections of the colorectal crypts.

Figure 1. Flow Diagram of a Trial of Supplemental Calcium and Vitamin D₃, Alone and in Combination vs. Placebo over Six Months on Markers of Apoptosis in the Normal Colorectal Mucosa

Figure 2. Distribution of Bax Staining Densities along Normal Colorectal Crypts by Treatment Group at Baseline and Follow-Up. A, Placebo Group. B, Calcium Group. C, Vitamin D Group. D, Calcium + Vitamin D Group

Figure 3. Distribution of Bcl-2 Staining Densities along Normal Colorectal Crypts by Treatment Group at Baseline and Follow-Up. A, Placebo Group. B, Calcium Group. C, Vitamin D Group. D, Calcium + Vitamin D Group