Inflammation Modulation by Vitamin D and Calcium in the Morphologically Normal Colorectal Mucosa of Patients with Colorectal Adenoma in a Clinical Trial.

Increased COX-2 and decreased 15-hydroxyprostaglandin dehydrogenase (15-HPGD) expression promote prostaglandin-mediated inflammation and colorectal carcinogenesis. Experimental studies suggest that vitamin D and calcium may inhibit these pathways, but their effects on colorectal tissue COX-2 and 15-HPGD expression in humans are unknown. We tested the effects of supplemental vitamin D (1,000 IU/day) and/or calcium (1,200 mg/day) on COX-2 and 15-HPGD expression in the morphologically normal rectal mucosa from 62 paients with colorectal adenoma in a placebo-controlled chemoprevention trial. We measured biomarker expression using automated IHC and quantitative image analysis at baseline and 1-year follow-up, and assessed treatment effects using mixed linear models. The primary outcome was the COX-2/15-HPGD expression ratio, because these enzymes function as physiologic antagonists. After 1 year of treatment, the mean COX-2/15-HPGD expression ratio in full-length crypts proportionately decreased 47% in the vitamin D group (P = 0.001), 46% in the calcium group (P = 0.002), and 34% in the calcium + vitamin D group (P = 0.03), relative to the placebo group. Among individuals with the functional vitamin D-binding protein isoform DBP2 (GC rs4588*A), the COX-2/15-HPDG ratio decreased 70% (P = 0.0006), 75% (P = 0.0002), and 60% (P = 0.006) in the vitamin D, calcium, and combined supplementation groups, respectively, relative to placebo. These results show that vitamin D and calcium favorably modulate the balance of expression of COX-2 and 15-HPGD-biomarkers of inflammation that are strongly linked to colorectal carcinogenesis-in the normal-appearing colorectal mucosa of patients with colorectal adenoma (perhaps especially those with the DBP2 isoform). PREVENTION RELEVANCE: Supplemental calcium and vitamin D reduce indicators of cancer-promoting inflammation in normal colorectal tissue in humans, thus furthering our understanding of how they may help prevent colorectal cancer.

Effects of Supplemental Calcium and Vitamin D on Circulating Biomarkers of Gut Barrier Function in Patients with Colon Adenoma: A Randomized Clinical Trial.

Gut barrier dysfunction promotes chronic inflammation, contributing to several gastrointestinal diseases, including colorectal cancer. Preliminary evidence suggests that vitamin D and calcium could prevent colorectal carcinogenesis, in part, by influencing gut barrier function. However, relevant human data are scarce. We tested the effects of supplemental calcium (1,200 mg/day) and/or vitamin D3 (1,000 IU/day) on circulating concentrations of biomarkers of gut permeability (anti-flagellin and anti-lipopolysaccharide IgA and IgG, measured via ELISA) from baseline to 1 and 3 or 5 years postbaseline among 175 patients with colorectal adenoma in a randomized, double-blinded, placebo-controlled clinical trial. We also assessed factors associated with baseline concentrations of these biomarkers. We found no appreciable effects of supplemental vitamin D3 and/or calcium on individual or aggregate biomarkers of gut permeability. At baseline, a combined permeability score (the summed concentrations of all four biomarkers) was 14% lower among women (P = 0.01) and 10% higher among those who consumed >1 serving per day of red or processed meats relative to those who consumed none (P trend = 0.03). The permeability score was estimated to be 49% higher among participants with a body mass index (BMI) > 35 kg/m2 relative to those with a BMI < 22.5 kg/m2 (P trend = 0.17). Our results suggest that daily supplemental vitamin D3 and/or calcium may not modify circulating concentrations of gut permeability biomarkers within 1 or 3-5 years, but support continued investigation of modifiable factors, such as diet and excess adiposity, that could affect gut permeability. PREVENTION RELEVANCE: Calcium and vitamin D may be involved in regulating and maintaining the integrity of the intestinal mucosal barrier, the dysfunction of which results in exposure of the host to luminal bacteria, endotoxins, and antigens leading to potentially cancer-promoting endotoxemia and chronic colon inflammation. While our results suggest that daily supplementation with these chemopreventive agents does not modify circulating concentrations of gut permeability biomarkers, they support continued investigation of other potential modifiable factors, such as diet and excess adiposity, that could alter gut barrier function, to inform the development of treatable biomarkers of risk for colorectal neoplasms and effective colon cancer preventive strategies.

Effects of supplemental calcium and vitamin D on tight-junction proteins and mucin-12 expression in the normal rectal mucosa of colorectal adenoma patients.

The physical gut barrier, comprised of a thick mucus layer and the epithelium, plays an important role in defense against microbes and foreign antigens. Calcium and vitamin D may be involved in maintaining the integrity of the intestinal mucosal barrier, the dysfunction of which may lead to endotoxemia and inflammation, and contribute to colorectal carcinogenesis. We investigated supplemental calcium (1200 mg, daily) and/or vitamin D3 (1000 IU daily) effects on intestinal barrier function-related biomarkers in a subset of 105 participants from a large colorectal adenoma recurrence chemoprevention clinical trial. We assessed expression of the tight junction proteins claudin-1 (CLDN1), occludin (OCLD), and mucin-12 (MUC12) in the normal-appearing colorectal mucosa using standardized, automated immunohistochemistry and quantitative image analysis. Following 1 year of treatment, in the calcium relative to the no calcium group, the CLDN1, OCLD, and MUC12 expression increased by 14% (P = 0.17), 23% (P = 0.11), and 22% (P = 0.07), respectively. In secondary analyses, the estimated calcium treatment effects were greater among participants with baseline serum 25-OH-vitamin D concentrations below the median value of 22.69 ng/mL (CLDN1: 29%, P = 0.04; OCLD: 36%, P = 0.06; MUC12: 35%, P = 0.05). There were no biomarker expression changes in the vitamin D3 alone group; however, modest increases were found in the combined calcium/vitamin D3 group. At baseline, obesity, history of a sessile-serrated adenoma, colorectal MIB-1/Ki-67 expression, and a family history of colorectal cancer were associated with CLDN1, OCLD, and MUC12 expression. Our study supports continued investigation of factors that could affect intestinal mucosal barrier integrity relevant to colorectal carcinogenesis.

Effects of vitamin D and calcium on expression of MSH2 and transforming growth factors in normal-appearing colorectal mucosa of sporadic colorectal adenoma patients: A randomized clinical trial.

Abnormal expression of the DNA mismatch repair protein MSH2 and autocrine/paracrine transforming growth factors TGFα (growth promoter) and TGFß1 (growth inhibitor) is common during colorectal carcinogenesis. To estimate vitamin D and calcium effects on these biomarkers in the normal-appearing colorectal mucosa of sporadic colorectal adenoma patients, we conducted a pilot, randomized, double-blinded, placebo-controlled, modified 2 × 2 factorial chemoprevention clinical trial (N = 104) of supplemental vitamin D3 (1000 IU daily) and calcium (1200 mg daily), alone and in combination, versus placebo over 1 year. The expression of the three biomarkers and Ki-67/mib-1 in colorectal crypts in biopsies of normal-appearing rectal mucosa were detected using automated immunohistochemistry and quantified using image analysis. In the vitamin D3 and vitamin D3 plus calcium groups, relative to their reference groups, in the upper 40% (differentiation zone) of crypts, it was estimated that, respectively, the MSH2/mib-1 ratio increased by 47% (P = 0.14) and 62% (P = 0.08), TGFß1 expression increased by 41% (P = 0.25) and 78% (P = 0.14), and the TGFα/TGFß1 ratio decreased by 25% (P = 0.31) and 44% (P = 0.13). Although not statistically significant, these results support further research into (i) whether supplemental vitamin D3 , alone or in combination with calcium, may increase DNA mismatch repair relative to proliferation, increase TGFß1 expression, and decrease autocrine/paracrine growth promotion relative to growth inhibition in the colorectal epithelium, all hypothesized to reduce risk for colorectal carcinogenesis; and (ii) the expression of MSH2 relative to mib-1, TGFß1 alone, and TGFα relative to TGFß1 in the normal-appearing rectal mucosa as potential modifiable, pre-neoplastic markers of risk for colorectal neoplasms.

Effects of supplemental vitamin D and calcium on markers of proliferation, differentiation, and apoptosis in the normal colorectal mucosa of colorectal adenoma patients.

To clarify the roles of vitamin D and calcium as potential chemopreventive agents against colorectal cancer in humans, and to develop "treatable", pre-neoplastic, phenotypic biomarkers of risk for colorectal neoplasms, we estimated the effects of supplemental vitamin D3 (1,000 IU/day [25 µg/day]) and calcium (1,200 mg/day), alone and in combination, on biomarkers of proliferation (mib-1), differentiation (p21), and apoptosis (bax [apoptosis-promoting] and bcl-2 [apoptosis-inhibiting]), in the normal-appearing rectal mucosa in a subsample of participants (n = 104) in a larger randomized, double-blind, placebo-controlled clinical trial among colorectal adenoma patients. The biomarkers were measured in rectal biopsies at baseline and after one year of follow up, using automated immunohistochemistry and quantitative image analysis. In the vitamin D plus calcium group relative to control, in the crypt differentiation zone (upper 40% of crypts), mib-1 expression decreased 24% (P = 0.28); p21 expression alone and relative to mib-1 expression increased 29% (P = 0.06) and 73% (P = 0.06), respectively; and bax expression relative to mib-1 expression increased 58% (P = 0.21). The estimated vitamin D alone treatment effects were similar but of lesser magnitudes, and those for calcium alone were mixed. All estimated treatment effects on bcl-2 expression were close to the null. These pilot study results support further investigation of whether 1) vitamin D and calcium promote colorectal epithelial cell differentiation, reduce proliferation, and promote apoptosis in the normal-appearing human colorectal mucosa, 2) vitamin D and calcium act as chemopreventive agents against colorectal neoplasms, and 3) mib-1, p21, and bax are potential "treatable", pre-neoplastic, biomarkers of risk for colorectal neoplasms.

Effects of Supplemental Calcium and Vitamin D on Expression of Toll-Like Receptors and Phospho-IKKα/ß in the Normal Rectal Mucosa of Colorectal Adenoma Patients.

Chronic inflammation in the colorectum, a significant contributor to colorectal carcinogenesis, can be triggered by the activation of proinflammatory signaling pathways such as those initiated by Toll-like receptors (TLR) and nuclear factor κB (NF-κB). Although experimental evidence supports calcium and vitamin D potentially modifying these proinflammatory pathways in the colorectum, human data in these regards are scarce. We investigated supplemental calcium (1,200 mg daily) and/or vitamin D3 (1,000 IU daily) effects on inflammatory signaling pathway-related biomarkers in a subset of 105 participants from a colorectal adenoma recurrence chemoprevention clinical trial. We assessed expression of TLR4 and TLR5, which recognize the bacterial components lipopolysaccharides and flagellin, respectively, and phospho-IKKα/ß (pIKKα/ß), a biomarker of inflammation, in the normal-appearing rectal crypt epithelium and stroma using standardized, automated immunohistochemistry and quantitative image analysis. Following 1 year of treatment, TLR4, TLR5, and pIKKα/ß expression in the rectal mucosa did not statistically significantly change with vitamin D or calcium supplementation, taken alone or in combination. Several baseline participant characteristics, including body mass index, history of sessile serrated adenomas, high red/processed meat intake, and high levels of rectal epithelial cell proliferation (as measured by MIB-1/Ki-67), were associated with higher baseline expression of TLRs or pIKKα/ß. Our findings suggest that vitamin D and calcium may have no substantial effect on the investigated biomarkers. However, several modifiable lifestyle factors may be associated with TLRs and pIKKα/ß expression in the normal rectal mucosa, supporting their future investigation as potentially treatable, preneoplastic risk factors for colorectal neoplasms. Cancer Prev Res; 11(11); 707-16. ©2018 AACR.

Effects of supplemental calcium and vitamin D on the APC/ß-catenin pathway in the normal colorectal mucosa of colorectal adenoma patients.

APC/ß-catenin pathway malfunction is a common and early event in colorectal carcinogenesis. To assess calcium and vitamin D effects on the APC/ß-catenin pathway in the normal-appearing colorectal mucosa of sporadic colorectal adenoma patients, nested within a larger randomized, double-blind, placebo-controlled, partial 2 × 2 factorial chemoprevention clinical trial of supplemental calcium (1200 mg daily) and vitamin D (1000 IU daily), alone and in combination versus placebo, we assessed APC, ß-catenin, and E-cadherin expression in colon crypts in normal-appearing rectal mucosa biopsies from 104 participants at baseline and 1-yr follow up using standardized, automated immunohistochemistry and quantitative image analysis. For vitamin D versus no vitamin D, the ratio of APC expression to ß-catenin expression in the upper 40% (differentiation zone) of crypts (APC/ß-catenin score) increased by 28% (P = 0.02), for calcium versus no calcium it increased by 1% (P = 0.88), and for vitamin D + calcium versus calcium by 35% (P = 0.01). Total E-cadherin expression increased by 7% (P = 0.35) for vitamin D versus no vitamin D, 8% (P = 0.31) for calcium versus no calcium, and 12% (P = 0.21) for vitamin D + calcium versus calcium. These results support (i) that vitamin D, alone or in combination with calcium, may modify APC, ß-catenin, and E-cadherin expression in humans in directions hypothesized to reduce risk for colorectal neoplasms; (ii) vitamin D as a potential chemopreventive agent against colorectal neoplasms; and (iii) the potential of APC, ß-catenin, and E-cadherin expression as treatable, pre-neoplastic risk biomarkers for colorectal neoplasms. © 2016 Wiley Periodicals, Inc.

Race and Prevalence of Large Bowel Polyps Among the Low-Income and Uninsured in South Carolina.

BACKGROUND: Compared to whites, blacks have higher colorectal cancer incidence and mortality rates and are at greater risk for early-onset disease. The reasons for this racial disparity are poorly understood, but one contributing factor could be differences in access to high-quality screening and medical care. AIMS: The present study was carried out to assess whether a racial difference in prevalence of large bowel polyps persists within a poor and uninsured population (n = 233, 124 blacks, 91 whites, 18 other) undergoing screening colonoscopy. METHODS: Eligible patients were uninsured, asymptomatic, had no personal history of colorectal neoplasia, and were between the ages 45-64 years (blacks) or 50-64 years (whites, other). We examined the prevalence of any adenoma (conventional, serrated) and then difference in adenoma/polyp type by race and age categories. RESULTS: Prevalence for ≥1 adenoma was 37 % (95 % CI 31-43 %) for all races combined and 36 % in blacks <50 years, 38 % in blacks ≥50 years, and 35 % in whites. When stratified by race, blacks had a higher prevalence of large conventional proximal neoplasia (8 %) compared to whites (2 %) (p value = 0.06) but a lower prevalence of any serrated-like (blacks 18 %, whites 32 %; p value = 0.02) and sessile serrated adenomas/polyps (blacks 2 %, whites 8 % Chi-square p value; p = 0.05). CONCLUSIONS: Within this uninsured population, the overall prevalence of adenomas was high and nearly equal by race, but the racial differences observed between serrated and conventional polyp types emphasize the importance of taking polyp type into account in future research on this topic.

A Trial of Calcium and Vitamin D for the Prevention of Colorectal Adenomas.

BACKGROUND: Epidemiologic and preclinical data suggest that higher intake and serum levels of vitamin D and higher intake of calcium reduce the risk of colorectal neoplasia. To further study the chemopreventive potential of these nutrients, we conducted a randomized, double-blind, placebo-controlled trial of supplementation with vitamin D, calcium, or both for the prevention of colorectal adenomas. METHODS: We recruited patients with recently diagnosed adenomas and no known colorectal polyps remaining after complete colonoscopy. We randomly assigned 2259 participants to receive daily vitamin D3 (1000 IU), calcium as carbonate (1200 mg), both, or neither in a partial 2×2 factorial design. Women could elect to receive calcium plus random assignment to vitamin D or placebo. Follow-up colonoscopy was anticipated to be performed 3 or 5 years after the baseline examinations, according to the endoscopist's recommendation. The primary end point was adenomas diagnosed in the interval from randomization through the anticipated surveillance colonoscopy. RESULTS: Participants who were randomly assigned to receive vitamin D had a mean net increase in serum 25-hydroxyvitamin D levels of 7.83 ng per milliliter, relative to participants given placebo. Overall, 43% of participants had one or more adenomas diagnosed during follow-up. The adjusted risk ratios for recurrent adenomas were 0.99 (95% confidence interval [CI], 0.89 to 1.09) with vitamin D versus no vitamin D, 0.95 (95% CI, 0.85 to 1.06) with calcium versus no calcium, and 0.93 (95% CI, 0.80 to 1.08) with both agents versus neither agent. The findings for advanced adenomas were similar. There were few serious adverse events. CONCLUSIONS: Daily supplementation with vitamin D3 (1000 IU), calcium (1200 mg), or both after removal of colorectal adenomas did not significantly reduce the risk of recurrent colorectal adenomas over a period of 3 to 5 years. (Funded by the National Cancer Institute; ClinicalTrials.gov number, NCT00153816.).

Transforming growth factors and receptor as potential modifiable pre-neoplastic biomarkers of risk for colorectal neoplasms.

Increased colorectal epithelial cell proliferation is an early, common event in colorectal carcinogenesis. We conducted a pilot, colonoscopy-based case-control study (n = 49 cases, 154 controls) of incident, sporadic colorectal adenoma to investigate endogenous cell growth factors and receptor, as well as the balance of growth factors, as potential modifiable pre-neoplastic biomarkers of risk for colorectal neoplasms. We measured transforming growth factor alpha (TGFα), TGFß(1), and TGFß receptor II (TGFßRII) expression in normal-appearing mucosa from the rectum, sigmoid colon, and ascending colon using automated immunohistochemistry and quantitative image analysis. Diet and lifestyle were assessed via questionnaires. The mean ratio of rectal TGFα to TGFß(1) expression and mean rectal TGFα expression were, respectively, 110% (P = 0.02) and 49% (P = 0.04) higher in cases than in controls, and associated with a more than two-fold (OR 2.42, 95% CI 0.85-6.87) and a 62% (OR 1.62, 95% CI 0.63-4.19) higher risk of colorectal adenoma. TGFß(1) and TGFßRII expression were 6.7% (P = 0.75) and 7.2% (P = 0.49), respectively, lower in cases than in controls. The TGFα/TGFß(1) expression ratio was 105% higher among smokers than among non-smokers (P = 0.03). These preliminary data suggest that the balance of TGFα and TGFß(1) expression, and to a lesser extend TGFα alone, in the normal-appearing rectal mucosa may be directly associated with risk for incident, sporadic colorectal neoplasms, as well as with modifiable risk factors for colorectal neoplasms.

Markers of the APC/ß-catenin signaling pathway as potential treatable, preneoplastic biomarkers of risk for colorectal neoplasms.

BACKGROUND: Malfunctioning of the adenomatous polyposis coli (APC)/ß-catenin signaling pathway is both an early and common event in sporadic colorectal cancer. To assess the potential of APC/ß-catenin signaling pathway markers as treatable, preneoplastic biomarkers of risk for colorectal neoplasms, we conducted a pilot colonoscopy-based case-control study (51 cases and 154 controls) of incident, sporadic colorectal adenoma. METHODS: We evaluated APC, ß-catenin, and E-cadherin expression in normal mucosa from the rectum and ascending and sigmoid colon using automated immunohistochemical and quantitative image analysis. Diet, lifestyle, and medical history were assessed with validated questionnaires. RESULTS: In the normal rectal mucosa, the ratio of the proportion of APC expression in the upper 40% of crypts with total ß-catenin expression (APC/ß-catenin score) was 14.3% greater in controls than in cases [P = 0.02; OR, 0.40; 95% confidence interval (CI), 0.14-1.14]. Compared with controls, in cases, APC expression was 3.2% lower, ß-catenin expression was 3.0% higher, and E-cadherin expression was 0.7% lower; however, none of these differences were statistically significant. The APC/ß-catenin score statistically significantly differed according to categories of plausible risk factors for colorectal cancer [e.g., it was 17.7% higher among those with 25(OH) vitamin D(3) concentrations ≥ 27 ng/mL]. CONCLUSIONS: These preliminary data suggest that the combined expression of APC and ß-catenin in the normal rectal mucosa may be associated with risk for incident, sporadic colorectal neoplasms, as well as with modifiable risk factors for colorectal neoplasms. IMPACT: Our results may help advance the development of treatable, preneoplastic biomarkers of risk for colorectal neoplasms.

Colorectal mucosal expression of MSH2 as a potential biomarker of risk for colorectal neoplasms.

To characterize the expression of the mismatch repair gene MSH2 in normal colorectal crypts in humans and assess parameters of its expression as a potential modifiable biomarker of risk for colorectal neoplasms, we conducted a pilot, colonoscopy-based case-control study (51 cases and 154 controls) of incident, sporadic colorectal adenoma. Biopsies of normal-appearing rectal, sigmoid, and ascending colon mucosa were procured, immunohistochemically processed for MSH2 protein, and analyzed using custom quantitative image analysis procedures. MSH2 expression in adenoma cases was lower than in controls by 49% (P = 0.01) and 23% (P = 0.06) in the ascending colon and rectum, respectively, but not in the sigmoid colon. MSH2 expression in the rectum was 39% (P = 0.04) higher in subjects who regularly took a nonsteroidal anti-inflammatory drug than in those who did not, and it tended to be lower in those with adenomas in the right colon and those who had an adenoma with more advanced characteristics. These preliminary data suggest that lower MSH2 expression in the normal colonic mucosa, at least in the ascending colon and rectum, may be associated with increased risk of incident, sporadic colorectal adenoma as well as with modifiable risk factors for colorectal neoplasms, thus supporting further investigation of MSH2 expression as a potential modifiable biomarker of risk for colorectal neoplasms.

MutL-homolog 1 expression and risk of incident, sporadic colorectal adenoma: search for prospective biomarkers of risk for colorectal cancer.

To characterize the expression of the mismatch repair gene MutL-homolog 1 (MLH1) in normal colorectal crypts in humans, and assess parameters of its expression as a potential biomarker of risk for colorectal neoplasms, we conducted a pilot, colonoscopy-based case-control study (51 cases, 154 controls) of incident, sporadic colorectal adenoma. Biopsies of normal-appearing rectal, sigmoid, and ascending colon mucosa were procured, immunohistochemically processed for MLH1 protein, and analyzed using custom quantitative image analysis procedures. MLH1 expression in the ascending colon was, on average, 49% proportionally lower in cases than controls (P = 0.03), but there was little evidence for case-control differences in the rectum and sigmoid colon. In cases and controls, average MLH1 expression in the ascending colon tended to be lower with increased age [by 56% (P = 0.02) and 25% (P = 0.16), respectively, for those > or =55 years], and with a history of colorectal cancer in a first-degree relative (by 22% [P = 0.56] and 34% [P = 0.16], respectively). Among cases, but not controls, average MLH1 expression tended to be higher with current alcohol consumption, regular aspirin use, and higher total intakes of calcium, vitamin D, and folate. There was little indication of similar differences in the rectum. These preliminary data suggest that lower MLH1 expression in the normal colonic mucosa, at least in the ascending colon, may be associated with increased risk of incident, sporadic colorectal adenoma, as well as with modifiable risk factors for colorectal neoplasms, thus supporting further investigation of MLH1 expression as a potential "treatable" biomarker of risk for colorectal neoplasms.

TGF-alpha expression as a potential biomarker of risk within the normal-appearing colorectal mucosa of patients with and without incident sporadic adenoma.

BACKGROUND: Transforming growth factor-alpha (TGF-alpha), a stimulatory growth factor and member of the epidermal growth factor family, is a mediator of oncogenesis and malignant progression in colorectal carcinogenesis. Limited evidence suggests its utility as a growth-related biomarker of risk for colorectal cancer. METHODS: We measured expression of TGF-alpha in biopsies of normal-appearing colorectal mucosa using automated immunohistochemistry and quantitative image analysis in a subsample of 29 cases and 31 controls from a colonoscopy-based case-control study (n = 203) of biomarkers of risk for incident sporadic colorectal adenoma. Diet, lifestyle, and medical history were assessed with validated questionnaires. RESULTS: TGF-alpha expression in the rectum was 51% higher in cases compared with controls (P = 0.05) and statistically significantly associated with accepted risk factors for colorectal neoplasms (36% lower among nonsteroidal anti-inflammatory drug users, 49% lower among women using hormone replacement therapy, 79% higher among persons with a family history of colorectal cancer). CONCLUSIONS: TGF-alpha expression in the normal-appearing rectal mucosa shows promise as an early, potentially modifiable biomarker of risk for colorectal cancer.