Differential preventive activity of sulindac and atorvastatin in Apc+/Min-FCCCmice with or without colorectal adenomas

Abstract

Objective: The response of subjects to preventive intervention is heterogeneous. The goal of this study was to determine if the efficacy of a chemopreventive agent differs in non-tumour-bearing animals versus those with colorectal tumours. Sulindac and/or atorvastatin was administered to Apc^+/Min-FCCC mice with known tumour-bearing status at treatment initiation.

Design: Male mice (6-8 weeks old) underwent colonoscopy and received control chow or chow with sulindac (300 ppm), atorvastatin (100 ppm) or sulindac/atorvastatin. Tissues were collected from mice treated for 14 weeks (histopathology) or 7 days (gene expression). Cell cycle analyses were performed on SW480 colon carcinoma cells treated with sulindac, atorvastatin or both.

Results: The multiplicity of colorectal adenomas in untreated mice bearing tumours at baseline was 3.6-fold higher than that of mice that were tumour free at baseline (P=0.002). Atorvastatin completely inhibited the formation of microadenomas in mice that were tumour free at baseline (P=0.018) and altered the expression of genes associated with stem/progenitor cells. Treatment of tumour-bearing mice with sulindac/atorvastatin led to a 43% reduction in the multiplicity of colorectal adenomas versus untreated tumour-bearing mice (P=0.049). Sulindac/atorvastatin increased the expression of Hoxb13 and Rprm significantly, suggesting the importance of cell cycle regulation in tumour inhibition. Treatment of SW480 cells with sulindac/atorvastatin led to cell cycle arrest (G0/G1).

Conclusions: The tumour status of animals at treatment initiation dictates response to therapeutic intervention. Atorvastatin eliminated microadenomas in tumour-free mice. The tumour inhibition observed with Sul/Atorva in tumour-bearing mice was greater than that achieved with each agent.

Keywords: cancer prevention; colonoscopy; colorectal adenomas; familial adenomatous polyposis.

Figure 1

Representative colon lesions in Apc^+/Min-FCCC mice. (A) Images of adenomas protruding from the wall of the colon, obtained using a rigid bore endoscope. Forceps (1 mm in diameter) are included for size comparison. (B) Microadenoma consisting of three crypts (100× view). (C) Adenoma with characteristic irregular crypt structure (40× view). Inserts in (B) and (C) are respective high power views (400×). Images in (B) and (C) are of mice that completed the treatment regimen (14 weeks).

Figure 2

Multiplicity of intestinal adenomas in Apc^+/Min-FCCC mice following drug exposure. All evaluations were conducted after 14 weeks of drug treatment. (A) Multiplicity of gross small intestinal adenomas per treatment group, irrespective of tumour status at baseline. (B) Volume of histopathologically confirmed colon tumours by treatment group, irrespective of tumour status at baseline. (C) Multiplicity of colorectal adenomas in mice by treatment group, as defined in (A), stratified for the presence or absence of colon tumours at the time of treatment initiation (week 0). (D) Multiplicity of colorectal adenomas in all mice by treatment group (P>0.05), irrespective of tumour status at baseline. Results are expressed as the mean±SEM per group. The brackets denote group comparisons that achieved statistical significance using the Mann-Whitney test (tumour multiplicity and volume). Atorva, atorvastatin; Sul, sulindac; wk, week.

Figure 3

Effect of sulindac and atorvastatin on the cell cycle. (A) Heat map of cell cycle regulatory genes that are differentially expressed in untreated tumour-bearing mice versus those treated with Sul/Atorva. Neoplastic colonic epithelial cells were laser microdissected for microarray analyses. The intensity of the colour indicates the degree of upregulation (magenta) or downregulation (green) when the data are expressed as a ratio (Sul/Atorva vs control). Multiple listings of a gene reflect the analysis of several probes for the same gene. (B) Sul/Atorva induces cell cycle arrest. SW480 human colon carcinoma cells were treated with various doses of sulindac, atorvastatin or Sul/Atorva for 48 hours, stained with propidium iodide and analysed by flow cytometry for DNA content. Histograms were generated using FlowJo software. Values from a representative experiment are presented, with similar results obtained in two independent experiments. Atorva, atorvastatin; DMSO, dimethyl sulfoxide; Sul, sulindac.

Figure 4

Multiplicity of colonic microadenomas and colon tumour incidence in Apc^+/Min-FCCC mice, following drug exposure. (A) Multiplicity of colorectal microadenomas (≤4 crypts) in mice by treatment group (as defined in (B)), stratified for the presence or absence of colon tumours at the time of treatment initiation (week 0). (B) Incidence of colon tumours (adenomas+microadenomas) per treatment group among mice that were tumour free at baseline. All colon tumours were confirmed histopathologically. Results are expressed as the mean±SEM per group. The brackets denote group comparisons that achieved statistical significance using the Mann-Whitney test (tumour multiplicity) and the two-sample test of proportions (tumour incidence). Atorva, atorvastatin; Sul, sulindac; wk, week.

Figure 5

Heat map of stem/progenitor cell genes differentially expressed in atorvastatin-treated, tumour-free versus tumour-bearing mice. Normal colonic epithelial cells were laser microdissected from crypts for microarray analysis. The intensity of the colour indicates the degree of upregulation (magenta) or downregulation (green) when the level of expression in tumour-free versus tumour-bearing mice is expressed as a ratio.