Nutr Res Pract.  2012 Oct;6(5):396-404.

Inhibitory effects of calcium against intestinal cancer in human colon cancer cells and ApcMin/+ mice

• Ju J
• Kwak Y
• Hao X
• Yang CS

Abstract

The aim of the study was to investigate the inhibitory effects of calcium against intestinal cancer in vitro and in vivo. We first investigated the effects of calcium treatment in HCT116 and HT29 human colon cancer cells. At the concentration range of 0.8-2.4 mM, calcium significantly inhibited cell growth (by 9-29%), attachment (by 12-26%), invasion (by 15-31%), and migration (by 19-61%). An immunofluorescence microscope analysis showed that the treatment with calcium (1.6 mM) for 24 h increased plasma membrane beta-catenin but decreased nuclear beta-catenin levels in HT29 cells. We then investigated the effect of dietary calcium on intestinal tumorigenesis in ApcMin/+ mice. Mice received dietary treatment starting at 6 weeks of age for the consecutive 8 weeks. The basal control diet contained high-fat (20% mixed lipids by weight) and low-calcium (1.4 mg/g diet) to mimic the average Western diet, while the treatment diet contained an enriched level of calcium (5.2 mg calcium/g diet). The dietary calcium treatment decreased the total number of small intestinal tumors (by 31.4%; P < 0.05). The largest decrease was in tumors which were > or = 2 mm in diameter, showing a 75.6% inhibition in the small intestinal tumor multiplicity (P < 0.001). Immunohistochemical analysis showed significantly reduced nuclear staining of beta-catenin (expressed as nuclear positivity), but increased plasma membrane staining of beta-catenin, in the adenomas from the calcium-treated groups in comparison to those from the control group (P < 0.001). These results demonstrate intestinal cancer inhibitory effects of calcium both in human colon cancer cells and Apc Min/+ mice. The decreased beta-catenin nuclear localization caused by the calcium treatment may contribute to the inhibitory action.

Keyword

Calcium; human colon cancer cells; ApcMin/+ mice; beta-catenin

MeSH Terms

Adenoma
Animals
beta Catenin
Calcium
Calcium, Dietary
Cell Membrane
Cell Transformation, Neoplastic
Colon
Colonic Neoplasms
Diet
Fluorescent Antibody Technique
HT29 Cells
Humans
Hydrazines
Intestinal Neoplasms
Mice
Calcium
Calcium, Dietary
Hydrazines
beta Catenin

Figure

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  Fig. 1 Effects of the calcium treatment on cell growth, attachment and invasion in human colon cancer cells. Percentage of viable cells after the treatment of HCT116 cells with 0.8, 1.2, and 2.0 mM calcium for 72 h (A) and HT29 cells with 0.4, 0.8, 1.6 and 2.4 mM calcium for 48 h in serum complete medium (B). Percent of attached cells on the bottom of culture plate after the treatment of HCT116 cells with 0.8, 1.2, and 2.0 mM calcium in serum complete medium (C). Percentage of invaded cells to the outer chamber containing serum complete media from the inner chamber containing serum free media after the treatment of HCT116 cells with 0.4, 0.8, 1.2, 1.6 and 2.0 mM calcium for 48 h (D). The values shown are mean ± SE. Different letters (a-d) indicate statistical difference by one-way ANOVA followed by Tukey test (P < 0.05).

• 

  Fig. 2 Effects of the calcium treatment on cell migration in human colon cancer cells. Percentage of wound closure after the treatment of HCT116 and HT29 cells with 0.8, 1.2, and 2.0 mM calcium for 24 h (in the case of HCT116 cells) and 48 h (in the case of HT29 cells) in serum complete medium (A). The values shown are mean ± SE. Different letters (a, b) indicate a statistical difference by one-way ANOVA followed by Tukey test (P < 0.05). Representative pictures of the wound closure in HT29 cells with or without 2.0 mM calcium for 48 h (B). Dying cells were observed around the edge of wound in calcium-treated HT29 cells at the 48 h time points, but not in the control cells (C).

• 

  Fig. 3 Effects of the calcium treatment on the localization of β-catenin in HT29 human colon cancer cells. Fluorescence microscopy analyses of cells in the absence (A) or presence of 1.6 mM extracellular calcium for 24 h (B). The yellow boxes in the panels A and B were magnified and shown in the panels C and D, respectively. Red and blue immunofluorescences are due to fluorescein isothiocyanate- and 4V,6-diamidino-2-phenylindole-conjugated secondary antibodies used for the staining of β-catenin and nucleus, respectively. Results are representatives of two independent experiments with consistent results.

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  Fig. 4 Effects of the dietary calcium treatment for 8 weeks on the localization of β-catenin and E-cadherin in the small intestinal adenomas from ApcMin/+ mice. Strong plasma membrane staining of β-catenin was observed in the normal mucosa (indicated as "N") from both untreated control (A) and calcium-treated mice (B). Adenomas (indicated as "T") from the control mice showed strong nuclear and cytoplasmic β-catenin staining (A). Treatment with calcium reduced the level of nuclear staining but increased the level of membrane staining in the adenomas (B). Strong plasma membrane staining of E-cadherin was observed in normal mucosa from both untreated control (C) and calcium-treated mice (D). The treatment with calcium increased the plasma membrane staining levels of E-cadherin in adenomas (D).

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