Dietary pectin and calcium inhibit colonic proliferation in vivo by differing mechanisms

Abstract

Diet plays an important role in promoting and/or preventing colon cancer; however, the effects of specific nutrients remain uncertain because of the difficulties in correlating epidemiological and basic observations. Transmissible murine colonic hyperplasia (TMCH) induced by Citrobacter rodentium, causes significant hyperproliferation and hyperplasia in the mouse distal colon and increases the risk of subsequent neoplasia. We have recently shown that TMCH is associated with an increased abundance of cellular beta-catenin and its nuclear translocation coupled with up-regulation of its downstream targets, c-myc and cyclin D1. In this study, we examined the effects of two putatively protective nutrients, calcium and soluble fibre pectin, on molecular events linked to proliferation in the colonic epithelium during TMCH. Dietary intervention incorporating changes in calcium [high (1.0%) and low (0.1%)] and alterations in fibre content (6% pectin and fibre-free) were compared with the standard AIN-93 diet (0.5% calcium, 5% cellulose), followed by histomorphometry and immunochemical assessment of potential oncogenes. Dietary interventions did not alter the time course of Citrobacter infection. Both 1.0% calcium and 6% pectin diet inhibited increases in proliferation and crypt length typically seen in TMCH. Neither the low calcium nor fibre-free diets had significant effect. Pectin diet blocked increases in cellular beta-catenin, cyclin D1 and c-myc levels associated with TMCH by 70%, whereas neither high nor low calcium diet had significant effect on these molecules. Diets supplemented with either calcium or pectin therefore, exert anti-proliferative effects in mouse distal colon involving different molecular pathways. TMCH is thus a diet-sensitive model for examining the effect of specific nutrients on molecular characteristics of the pre-neoplastic colonic epithelium.

Figure 1

Effects of TMCH and diets on isolated crypts. Colonic crypts from distal colon of normal mice, Citrobacter‐infected mice at day 12 TMCH, Citrobacter‐infected mice on either a 6% pectin diet or a high‐calcium diet, were isolated as described in METHODS (crypts are marked with arrows). TMCH crypts essentially doubled in length compared with normal mice; dietary interventions reversed this elongation. Detailed morphometry is presented in Table 2.

Figure 2

Effect of differing diets on weight gain. Animals from five dietary treatment groups were weighed on day 3 after infection with Citrobacter rodentium, the time of the change in diets and weighed on day 10 and prior to killing on day 12. There were no significant differences among groups.

Figure 3

Effect of dietary intervention on normal colon. Western blots performed in total crypt extracts prepared from uninfected mice on a standard AIN93, 6% Pectin or 1% Ca²⁺ diets, did not show any alterations in PCNA, β‐catenin or cyclin D1 expression.

Figure 4

Time course and effect of dietary intervention on intimin gene expression during TMCH. (a) Citrobacter rodentium‐encoded intimin gene expression was measured by RT–PCR, in distal colon from mice on a control diet. Intimin expression was absent in normal colon (lane 1). While day 9 TMCH colon (lane 2) expressed intimin, neither day 12 or day 15 TMCH colon expressed detectable intimin (lanes 3 and 4, respectively), indicating lack of bacterial presence at peak hyperplasia (day 12). To determine whether dietary interventions affected Citrobacter infectivity, intimin expression was measured either by RT–PCR (b), or northern blotting (c) of total RNA extracted from normal (lane 1), Citrobacter (lane 2), Citrobacter + 6% pectin (lane 3) or Citrobacter + 1% Ca²⁺ (lane 4)‐treated whole distal colon. Lane 5 in (b) is no cDNA control. Intimin expression was not affected by either high pectin or calcium diets (n = 2).

Figure 5

Effects of dietary intervention on cellular β‐catenin protein abundance. Western blots were performed in uninfected animals on a control diet (lane 1); Citrobacter‐infected animals on a control diet (lane 2); Citrobacter‐infected animals on a fibre‐free diet (lane 3); Citrobacter‐infected animals on a 0.1% calcium diet (lane 4); Citrobacter‐infected animals on a 1.0% calcium diet (lane 5); and Citrobacter infected animals on a 6% pectin diet. Results were normalized to actin. γ‐catenin, an adhesion molecule without oncogenic potential, was employed as a control. Results demonstrate a 70% inhibition of β‐catenin abundance comparing the pectin to the control diet. The high‐calcium diet was associated with a modest 15% inhibition of β‐catenin protein abundance. There was small decrease in γ‐catenin abundance (13–26%). This blot is representative of n = 3 groups of nine animals.

Figure 6

Effects of dietary interventions on cyclin D1 and c‐myc abundance. Western blots were performed to measure the effect of changes in fibre and calcium on the previously demonstrated changes in these molecules at day 12 in the TMCH colon. The expected increase in basal cyclin D1 on a control diet (lane 5) to day 12 TMCH (lane 6) was seen. There were no significant changes induced by fibre‐free (lane 1) or changes in calcium content (0.1% Ca²⁺, lane 2: 1.0% Ca²⁺, lane 3). In contrast, there was a major decrease in cyclin D1 abundance on the pectin diet (lane 4, 70% inhibition). A similar pattern was seen when c‐myc protein abundance was compared in fibre‐free (lane 1), 0.1% calcium (lane 2), 1.0% calcium (lane 3) and pectin (lane 4). The increase in c‐myc on the pectin diet was inhibited by 70% (n = 3 groups of nine animals).