Regulation of the metabolite profile by an APC gene mutation in colorectal cancer

Abstract

Mutation of the APC gene occurs during the early stages of colorectal cancer development. To obtain new insights into the mechanisms underlying the aberrant activation of the Wnt pathway that accompanies APC mutation, we carried out a gas chromatography-mass spectrometry-based semiquantitative metabolome analysis. In vitro experiments comparing SW480 cells expressing normal APC and truncated APC indicated that the levels of metabolites involved in the latter stages of the intracellular tricarboxylic acid cycle, including succinic acid, fumaric acid, and malic acid, were significantly higher in the SW480 cells expressing the truncated APC. In an in vivo study, we found that the levels of most amino acids were higher in the non-polyp tissues of APC(min/+) mice than in the normal tissues of the control mice and the polyp tissues of APC(min/+) mice. Ribitol, the levels of which were decreased in the polyp lesions of the APC(min/+) mice and the SW480 cells expressing the truncated APC, reduced the growth of SW480 cells with the APC mutation, but did not affect the growth of SW480 transfectants expressing full-length APC. The level of sarcosine was found to be significantly higher in the polyp tissues of APC(min/+) mice than in their non-polyp tissues and the normal tissues of the control mice, and the treatment of SW480 cells with 50 μM sarcosine resulted in a significant increase in their growth rate. These findings suggest that APC mutation causes changes in energetic metabolite pathways and that these alterations might be involved in the development of colorectal cancer.

Figure 1

Metabolomic profiling of APC ^min/+ mice and SW480 human epithelial colorectal cancer cells. (A) Venn diagram based on the metabolites detected in sera and tissues of APC ^min/+ mice, corresponding control mice, and SW480 cells. The circle graph represents the distribution of chemical classes for the metabolites detected in sera (B), tissues (C), and SW480 cells (D). Others include amines, miscellaneous, heterocyclic molecules, ketones, and aldehydes. The values in parentheses indicate the percentage of total.

Figure 2

Mutation of the APC gene causes the development of adenoma in the digestive tract. (A) SW480 colorectal cancer cells expressing truncated APC and SW480 transfectants expressing full‐length APC were subjected to immunohistochemical analysis with anti‐β‐catenin antibody. Representative images are shown. Green, β‐catenin. Magnification, ×200. (B) SW480 cells expressing truncated APC and SW480 transfectants expressing full‐length APC were subjected to Western blot analysis with anti‐APC antibody and anti‐β‐actin antibody. Representative images are shown. (C) Small intestinal tissue was collected from APC ^min/+ mice with non‐polyp and polyp lesions and C57BL/6J mice and subjected to H&E staining. Representative images are shown. Magnification, ×100.

Figure 3

Effects of target metabolites on the cell growth rates of SW480 colorectal cancer cells and SW480 transfectants. SW480 cells were grown at 5 × 10⁴/well in 96‐well plates for 24 h then exposed to the indicated concentrations of inositol (A), ribitol (B), 2‐hydroxybutyric acid (C), 3‐hydroxypropanoic acid (D), and sarcosine (E). After 24 h, the rate of cell growth was evaluated. Data are shown as the mean ± SD, n = 3–10. Statistical significance was analyzed using Student's t‐test, and a level of probability of 0.05 was used as the criterion of significance. *P < 0.05. L‐proline (0.6–1.8 mM), arabitol (0.1–10 mM), and isoleucine (0.6–1.8 mM) did not significantly affect the rate of cell growth (data not shown).