Iron Supplementation Increases Tumor Burden and Alters Protein Expression in a Mouse Model of Human Intestinal Cancer

Abstract

Iron supplements are widely consumed. However, excess iron may accelerate intestinal tumorigenesis. To determine the effect of excess iron on intestinal tumor burden and protein expression changes between tumor and normal tissues, Apc^Min/+ mice were fed control (adequate) and excess iron (45 and 450 mg iron/kg diet, respectively; n = 9/group) for 10 wk. Tumor burden was measured, and two-dimensional fluorescence difference gel electrophoresis was used to identify differentially expressed proteins in tumor and normal intestinal tissues. There was a significant increase (78.3%; p ≤ 0.05) in intestinal tumor burden (mm²/cm) with excess iron at wk 10. Of 980 analyzed protein spots, 69 differentially expressed (p ≤ 0.05) protein isoforms were identified, representing 55 genes. Of the isoforms, 56 differed (p ≤ 0.05) between tumor vs. normal tissues from the adequate iron group and 23 differed (p ≤ 0.05) between tumors from the adequate vs. excess iron. Differentially expressed proteins include those involved in cell integrity and adaptive response to reactive oxygen species (including, by gene ID: ANPEP, DPP7, ITGB1, PSMA1 HSPA5). Biochemical pathway analysis found that iron supplementation modulated four highly significant (p ≤ 0.05) functional networks. These findings enhance our understanding of interplay between dietary iron and intestinal tumorigenesis and may help develop more specific dietary guidelines regarding trace element intake.

Keywords: intestinal cancer; iron supplementation; micronutrient; mouse model; protein expression; tumor.

Figure 1

(A) Intestinal segment opened longitudinally and stained with methylene blue to show tumors. Bi-dimensional measurements are also shown (“um” = μm); cross-section (diameter) measurements (lines) overlay each tumor. Normal villi can be seen surrounding tumors. Bar in upper left corner = 1000 μm (1 mm). (B) Tumor number (total/intestines) and tumor burden (mm²/cm) in intestines of mice fed adequate (AFe) and excess (High Fe) iron, at wk 10. (Black line represents tumor number and red line tumor burden; values are mean ± SEM for each treatment group).

Figure 2

(A) Overlay of 2D-DIGE fluorescence gel images, (B) individually captured fluorescence gel images using excitation/emission wavelengths of 488/520 nm, 532/580 nm, and 633/670 nm for Cy2 (internal standard), Cy3 (tumor tissue; adequate iron diet), and Cy5 (tumor tissue; excess iron diet), respectively, and (C) the accompanying 2D deep purple-labeled map of the intestinal tissue proteome, with spots of interest.

Figure 3

(A,B) Sample images of the 2D fluorescence difference gel electrophoresis (2D-DIGE) Cy3 and Cy5 labeled proteins from intestinal tumor tissues from adequate vs. excess iron, respectively, to illustrate differential protein expression. Circles show sample protein spots (expression) that either significantly (p ≤ 0.05) increased (1 and 2) or did not change (3) in tumor tissue with excess iron.

Figure 4

Two-dimensional (deep purple-labeled) protein spot map shown in Figure 2C showing orientation of the pH gradients on the horizontal axes, from pH 3 (left) to 10 (right), with approximate molecular mass ranges indicated along the vertical axes from 10 (near bottom) to 200 kDa (near top).

Figure 5

(A) Key differentially expressed (p ≤ 0.05) proteins in (A) tumor vs. adjacent non-tumor intestinal tissues from the adequate iron diet group and (B) tumor tissues from the excess vs. adequate iron diet groups. Proteins are presented by gene name. Fold-change expression differences are shown.

Figure 5

(A) Key differentially expressed (p ≤ 0.05) proteins in (A) tumor vs. adjacent non-tumor intestinal tissues from the adequate iron diet group and (B) tumor tissues from the excess vs. adequate iron diet groups. Proteins are presented by gene name. Fold-change expression differences are shown.

Figure 6

Protein networks of intestinal tumor tissues from adequate (A) vs. excess iron (B) revealed by pathway analysis. Solid lines indicate direct interaction and dashed lines indicate indirect interaction (protein abbreviations and identities are defined and described, respectively, in Table 2). An asterisk indicates that the protein is involved in cell membrane integrity or substrate transport. The intensity of red and green molecule colors indicates the degree of up-or-down-regulation, respectively. Myc- and p53- related networks (red circles) are associated with functions implemented in cancer and cell death. Select key differentially expressed protein names are shown.

Figure 6

Protein networks of intestinal tumor tissues from adequate (A) vs. excess iron (B) revealed by pathway analysis. Solid lines indicate direct interaction and dashed lines indicate indirect interaction (protein abbreviations and identities are defined and described, respectively, in Table 2). An asterisk indicates that the protein is involved in cell membrane integrity or substrate transport. The intensity of red and green molecule colors indicates the degree of up-or-down-regulation, respectively. Myc- and p53- related networks (red circles) are associated with functions implemented in cancer and cell death. Select key differentially expressed protein names are shown.