Suppression of intestinal carcinogenesis in Apc-mutant mice by limonin

Abstract

Limonoids in citrus fruits are known to possess multiple biological functions, such as anti-proliferative functions in human cancer cell lines. Therefore, we aimed to investigate the suppressive effect of limonin on intestinal polyp development in Apc-mutant Min mice. Five-week-old female Min mice were fed a basal diet or a diet containing 250 or 500 ppm limonin for 8 weeks. The total number of polyps in mice treated with 500 ppm limonin decreased to 74% of the untreated control value. Neoplastic cell proliferation in the polyp parts was assessed by counting PCNA positive cells, and a tendency of reduction was obtained by limonin treatment. Moreover, expression levels of c-Myc and MCP-1 mRNA in the polyp part were reduced by administration of limonin. We finally confirmed the effects of limonin on β-catenin signaling, and found limonin significantly inhibited T-cell factor/lymphocyte enhancer factor-dependent transcriptional activity in a dose-dependent manner in the Caco-2 human colon cancer cell line. Our results suggest that limonin might be a candidate chemopreventive agent against intestinal carcinogenesis.

Keywords: Min mice; Tcf/Lef; c-Myc; colon cancer; limonin.

Fig. 1

Effects of limonin on the size distribution of intestinal polyps in Min mice. Min mice were fed a basal diet (black filled box), 250 ppm (gray filled box) and 500 ppm (open box) limonin containing diet for 8 weeks. The number of polyps per mouse in each size class is given as a mean. *p<0.05.

Fig. 2

Change of ratio of PCNA and nuclear β-catenin accumulation in the intestinal polyp by limonin. Immunohistochemical staining for PCNA and β-catenin in intestinal polyp from Min mice fed a basal diet or limonin containing diet. Calculations of the ratio for PCNA (A), nuclear β-catenin accumulation (B) are described in Materials and Methods section. Bars indicate SD.

Fig. 3

Changes of inflammation- or proliferation-related factors in intestinal non-polyp and/or polyp parts of Min mice. Quantitative real-time PCR analysis were performed to determine c-Myc (A), COX-2 (B), MCP-1 (C), VCAM-1 (D) mRNA expression levels in the non-polyp or polyp parts of Min mice with or without 500 ppm limonin. Data are normalized with GAPDH expression level. Each expression levels in non-polyp parts of untreated group are set as 1. Data are mean ± SD, n = 6. **p<0.001, *p<0.05 vs 0 ppm.

Fig. 4

Effects of limonin treatment on Tcf/Lef-dependent transcriptional activity in human colon cancer cells. Caco-2 cells were cultured in medium containing with the indicated dose of limonin. Relative Tcf/Lef-dependent transcriptional activities are plotted as the ratio of the un-stimulated control value. Data are mean ± SD, n = 6. *p<0.001 vs 0 µM. Similar results were obtained from three separate experiments.