These data provide evidence for a substantial increase in cell differentiation, as indicated by increased expression of p21, in the normal colorectal epithelium of sporadic adenoma patients in response to vitamin D3 or calcium supplementation and, thus, are consistent with the hypothesis that increased levels of circulating vitamin D or a higher intake of calcium may reduce risk for colorectal neoplasms. Our data also suggest that vitamin D3 may have a slightly greater effect than calcium on p21 expression, and vitamin D combined with calcium may have a lesser treatment effect than either calcium or vitamin D alone on p21. Furthermore, the data provide no evidence that the overall colorectal epithelial cell proliferation rate, as indicated by the expression of short- and long-term markers of proliferation in the entire colorectal crypt, can be reduced by calcium and vitamin D, alone or in combination. However, our data suggested that calcium combined with vitamin D may shift downwards (“normalize”) the distribution of proliferating cells in the colorectal crypts as indicated by the expression of a long-, but not short-term marker of cell proliferation.
, a cyclin-dependent kinase inhibitor used in this study as a marker of differentiation, is a potent inducer of differentiation in intestinal colonocytes (10
), and its expression is known to be downregulated during the early stages of colon tumorigenesis (10
). p21 was also reported to participate in cell cycle regulation (9
) and control of DNA methylation (26
), and to interact with regulatory proteins, among which is calmodulin (27
). As was found in colon cancer cells in vitro
), we hypothesized that vitamin D and calcium would increase p21 expression in the normal human colorectal epithelium in vivo
. The plausibility of this hypothesis is supported by the fact that the p21 gene is a primary 1,25-(OH)2
-responsive gene with at least three vitamin D response element (VDRE)-containing regions within its promoter (33
); and that calcium, through the calcium-sensing receptor (CaSR), promotes differentiation in colorectal epithelial cells (31
). However, there is little literature regarding direct regulation of p21 by calcium, but there is some evidence that extracellular calcium activates protein kinase C, which is associated with the differential induction of p21 in the intestinal epithelium (3
). Also, an intracellular calcium gradient along the colon crypt that coincides with the differentiation compartment may modulate differentiation of the colonocytes, thus, regulating p21 expression (34
). As hypothesized, we observed the largest increase in p21 expression in the vitamin D group, and to a lesser extent in the calcium group; however, we found only a relatively small increase in the calcium plus vitamin D group, and a statistically significant antagonistic interaction between the two treatments. There are several possible explanations for the latter finding, including the possibility that the observed treatment effect in the calcium plus vitamin D group may have been due to chance, or that the two agents may have attenuated the effects of either alone. One animal study (35
) found that calcium and vitamin D separately are more potent inhibitors of colon tumorigenesis than when combined. However, several other animal studies that investigated the combined effect of calcium and vitamin D reported stronger effects with vitamin D and calcium combined (36
); and the results of a large adenoma recurrence trial suggested that vitamin D enhanced the chemopreventive effect of calcium (38
). Thus, the combined effect of calcium and vitamin D on colon crypt epithelial cell differentiation as indicated by p21 expression is not clear and a larger more definitive study is needed to clarify it.
No previous human studies tested the effect of calcium and/or vitamin D supplementation on p21 expression in the normal colorectal mucosa, but three small studies (15
) investigated the effects of these agents or low fat dairy foods on other markers of differentiation (acidic mucins and/or cytokeratin AE1) in the normal colorectal mucosa with inconsistent results. Two small studies found no changes in the normal rectal crypt differentiation markers after supplementation with calcium and vitamin D3
), or with calcium or low fat dairy foods (16
); but a third, larger (N=70), randomized, placebo-controlled trial reported significant changes in differentiation markers after supplementation with low fat dairy foods, which are rich in calcium and vitamin D, but contain other components that may also exert prodifferentiative effects (21
). Taken altogether, the results of the present and past studies combined with the biological evidence suggest that calcium and vitamin D induce differentiation in the normal human colorectal mucosa, and that expression of p21 may be a more suitable biomarker of differentiation than other currently investigated markers.
Unlike other studies, we used two different markers of proliferation, hTERT and MIB-1, detected by immunohistochemical methods. MIB-1/Ki-67 is expressed in all cells not in G0
phase of the cell cycle (12
); and hTERT protein, a catalytic subunit of telomerase, which functions to regenerate telomeres on the ends of chromosomes, is expressed in almost all human cancers and some normal proliferative epithelial cells such as in the colorectal crypt base (11
). We hypothesized that hTERT expression in colorectal crypts better reflects average, long term proliferative activity than do “snapshot” proliferative indicators, such as the S-phase marker MIB-1, which demonstrate rapid, large responses to short term physiologic stimuli. Biological evidence supports the growth-restraining actions of calcium and vitamin D on colorectal epithelial cells (3
), however few human studies tested the effect of vitamin D and calcium on cell proliferation in the colon.
There have been two large clinical trials of calcium and colorectal epithelial cell proliferation (13
) as well as several smaller trials (reviewed in (22
), also (16
)). One of these trials (N=193) found no evidence for a reduction in the labeling index (LI), but a marked, statistically significant proportional decrease in the h
), but the second trial (N=333) (14
), with more methodological problems (22
), found no effect on either measure of cell proliferation. The findings from several smaller controlled studies were inconsistent, with some suggesting decreases in the LI and/or h
, and other studies indicating no change or statistically non-significant increases in the LI and/or h
. The results of the present study for the LI are consistent with those from the previously conducted large clinical trials (13
); and for the h
with one large clinical trial (13
) and several smaller clinical trials (reviewed in (22
), also (42
)). However, it must be emphasized that the present study was a pilot study with limited statistical power; thus, our findings may have been due to chance. Other possible explanations for our findings may have been the use of an antibody that may have low specificity detecting hTERT (11
); that the MIB-1 and/or hTERT markers may not be good biomarkers of cell proliferation in normal colorectal crypts; or that calcium may indeed have no substantial effect on colorectal cell proliferation in sporadic adenoma patients.
No published human studies tested the effect of vitamin D alone or combined with calcium on the hTERT or MIB-1 markers of proliferation, but one small randomized clinical trial (N=21) found a significantly decreased MIB-1 labeling index, but not the h
, in flat mucosa and resected polypoid tissue after 6-months supplementation with calcium (1,500 mg/day) plus vitamin D3
(400 IU/day) (15
). Contrary to the results of one study (15
), we did not find evidence for an effect of vitamin D alone or in combination with calcium on overall MIB-1 or hTERT labeling, but we did find a significant downward shift in hTERT expression in the calcium plus vitamin D group. However, as pointed out above, these findings may be due to chance, non-specific detection methods, or an insufficient vitamin D3
dose or duration.
Previous studies (44
) and our study found that the LI and h
are statistically independent variables, and other controlled trials testing calcium or other agents on cell proliferation rates found statistically significant reductions in the h,
but not the LI (13
). Therefore, the LI and h
may represent different biological aspects of colon tumorigenesis, and serve as independent markers of risk for colorectal neoplasia.
The present study was conducted to test the joint and separate effects of calcium and vitamin D on the individual components and aggregate profile of a molecular phenotype panel of biomarkers of risk for colorectal cancer, which includes biomarkers of APC and mismatch repair pathways, cell cycle events, and others. We previously reported a statistically significant effect of vitamin D on the pro-apoptotic marker Bax (17
), and analyses for other biomarkers in the panel are currently underway. Taken all together, the present and previously published data (13
) suggest that calcium and vitamin D may have stronger effects on cell differentiation and apoptosis than on proliferation; and that even relatively low dose vitamin D may have greater effects on colorectal epithelial cell differentiation and apoptosis than does high dose calcium alone or in combination with low dose vitamin D. However, larger, more definitive clinical studies are needed to confirm these results.
This study has several limitations. The most obvious limitation is the small sample size resulting in an increased role for chance in detecting or not detecting a treatment effect. The small size also did not allow us to conduct additional subgroup analyses. Another limitation is that, although human studies have found that cell proliferation rates observed in the rectal mucosa are correlated with those found throughout the colon (48
), animal studies found that calcium affects cell proliferation throughout the colon (50
), and one intervention trial found that calcium decreases the LI and h
in the rectum and sigmoid colon, but not in the descending colon (45
), there are insufficient data to assume that the effect of calcium is the same in the distal and proximal parts of the colon in humans. Furthermore, the effects of vitamin D alone or in combination with calcium on proliferation and differentiation in different parts of the colon (other than the rectum) are not clear, as there were no such studies in humans. Also, it is unknown whether vitamin D and/or calcium may affect human normal colon, adenoma, and cancer tissue differently. Another potential limitation of this study is that proliferation and differentiation markers are evidentially well-supported, but not proven biomarkers of risk for colorectal neoplasms. Therefore, this study cannot prove that because calcium and vitamin D substantially increase p21 expression and may shrink the proliferative zone in the colorectal crypts, they can reduce risk for colorectal neoplasms. The findings of this study may not be generalizable to other populations. Finally, there may be more specific methods and antibodies to detect telomerase expression in colorectal crypts (11
), and MIB-1 and hTERT may not adequately reflect cell proliferation rates in normal-appearing colorectal crypts.
The strengths of this study are that it is, to our knowledge, the first clinical trial of the effects of calcium and vitamin D3, alone and in combination on colorectal epithelial proliferation and differentiation in sporadic adenoma patients; the randomized, double-blind, placebo-controlled trial design; evaluation of both long- and short-term proliferation markers; high protocol adherence by study participants; automated biopsy processing and immunostaining procedures; the use of quantitative image analysis; and the strict quality control and consequent high scoring reliability of rectal biopsies.
In summary, these preliminary results from this pilot clinical trial indicate that calcium and vitamin D increase colorectal epithelial cell differentiation and may have relatively little, if any, effect on overall proliferation rates in the colorectal mucosa, but do not rule out a potential normalization of the proliferative zone in the colorectal crypts. This study suggests that p21 expression may be a treatable biomarker of risk for colorectal neoplasms and supports further investigation of calcium and vitamin D3 as chemopreventive agents against colorectal neoplasms.