A Multi-Mineral Intervention to Modulate Colonic Mucosal Protein Profile: Results from a 90-Day Trial in Human Subjects

Abstract

The overall goal of this study was to determine whether Aquamin®, a calcium-, magnesium-, trace element-rich, red algae-derived natural product, would alter the expression of proteins involved in growth-regulation and differentiation in colon. Thirty healthy human subjects (at risk for colorectal cancer) were enrolled in a three-arm, 90-day interventional trial. Aquamin® was compared to calcium alone and placebo. Before and after the interventional period, colonic biopsies were obtained. Biopsies were evaluated by immunohistology for expression of Ki67 (proliferation marker) and for CK20 and p21 (differentiation markers). Tandem mass tag-mass spectrometry-based detection was used to assess levels of multiple proteins. As compared to placebo or calcium, Aquamin® reduced the level of Ki67 expression and slightly increased CK20 expression. Increased p21 expression was observed with both calcium and Aquamin®. In proteomic screen, Aquamin® treatment resulted in many more proteins being upregulated (including pro-apoptotic, cytokeratins, cell-cell adhesion molecules, and components of the basement membrane) or downregulated (proliferation and nucleic acid metabolism) than placebo. Calcium alone also altered the expression of many of the same proteins but not to the same extent as Aquamin®. We conclude that daily Aquamin® ingestion alters protein expression profile in the colon that could be beneficial to colonic health.

Keywords: Aquamin®; biomarkers; calcium; colon cancer chemoprevention; minerals; proteomic analysis; trace elements.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Histological features of the colonic mucosa and proliferation expression. (A): Crypt Length. Values represent means and standard deviations based on measurement of crypt length in individual crypts in each colon biopsy (P:70 and 85, CA:2 and 71, AQ:74 and 96–number of pre and post crypts, respectively) per treatment group (10 subjects per group before and after treatment). (B): Ki67 expression quantitation. Percentage of Ki67-positive nuclei is presented in each group. Values represent means and standard deviations based on evaluation of individual crypts in each colon biopsy (P:106 and 81, CA:63 and 65, AQ:102 and 82–number of pre and post crypts respectively) per treatment group (10 subjects per group before and after 90-day intervention). Each dot represents an individual subject (A,B). (C): Histological appearance and Ki67-stained histological images of the colonic mucosa from a representative subject in each treatment group before and after the 90-day intervention. Scale bar = 100 µm.

Figure 2

Differentiation panel expression. (A): CK20 expression quantitation. CK20 expression is presented by CK20 stain positivity. Values represent means and standard deviations based on evaluation of individual crypts in each colon biopsy (P:106 and 81, CA:63 and 65, AQ:102 and 82–number of pre and post crypts respectively) and luminal surface per treatment group (10 subjects per group before and after treatment for 90 days). (B): p21 expression quantitation. Percentage of strong positive (2+ and 3+) nuclei is used to present p21 expression. Values represent means and standard deviations based on evaluation of individual crypts in each colon biopsy (P:106 and 78, CA:59 and 63, AQ:103 and 65–number of pre and post crypts respectively) and luminal epithelial cells per treatment group (10 subjects per group before and after treatment for 90 days). (C): Correlation of CK20 and p21 expressions in all 30 subjects. r = 0.3819; p (two-tailed) = 0.0373. Each dot represents an individual subject (A–C). (D): CK20-stained histological images of colonic mucosa from a representative subject of each treatment group before and after the 90-day intervention. Scale bar = 100 µm. (E): p21-stained histological images of colonic mucosa from a representative subject of each treatment group before and after the 90-day intervention. Scale bar = 50 µm.

Figure 3

Proteomic expression of colon mucosal biopsies and response to interventions. (A): Upregulated and downregulated proteins in each cohort at 1.5-fold. Each bar represents the number of proteins up-regulated (1.5-fold; ≤1% FDR) or down-regulated (0.67-fold; ≤1% FDR) over the 90-day course of treatment with each of the three interventions. On the right, Venn diagrams show the overlap of these upregulated and downregulated moieties to provide common proteins among 3 groups or between 2 groups or unique to an intervention. The list of these upregulated proteins is presented in Table S3. While downregulated proteins are presented in Table S4. (B): Upregulated and downregulated proteins in each cohort at 1.1-fold. Each bar represents the number of proteins up-regulated (1.1-fold; ≤1% FDR) or down-regulated (0.9-fold; ≤1% FDR) over the 90-day course of treatment with each of the three interventions. On the right, Venn diagrams show the overlap of these upregulated and downregulated moieties to provide common proteins among 3 groups or between 2 groups or unique to an intervention. (C): Differential proteomic expression of upregulated proteins (of all interventions at 1.5-fold; ≤1% FDR) is presented in a heatmap. These proteins are listed in Table S3. (D): Differential proteomic expression of downregulated proteins (of all interventions at 1.5-fold; ≤1% FDR) is presented in a heatmap. These proteins are listed in Table S4.

Figure 4

Upregulated proteins of interest. A fold-change value of each protein’s abundance ratio is presented in response to Aquamin® and calcium interventions by comparing these to placebo. This pooled proteomic analysis was based on n = 10 subjects in each cohort.

Figure 5

Downregulated proteins of interest. A fold-change value of each protein’s abundance ratio is presented in response to Aquamin® and calcium interventions by comparing these to placebo. This pooled proteomic analysis was based on n = 10 subjects in each cohort.