Cimetidine and Clobenpropit Attenuate Inflammation-Associated Colorectal Carcinogenesis in Male ICR Mice

Abstract

Histamine and histamine receptors (Hrhs) have been identified as critical molecules during inflammation and carcinogenesis. This study was conducted to determine the effects of Hrh1-Hrh3 antagonists on inflammation-associated colorectal carcinogenesis. Male ICR mice were treated with azoxymethane (AOM, 10 mg/kg bw, i.p.) and 1.5% dextran sodium sulfate (DSS, drinking water for 7 days) to induce colorectal carcinogenesis. The mice were then fed diets containing test chemical (500 ppm terfenadine, 500 ppm cimetidine or 10 ppm clobenpropit) for 15 weeks. At week 18, feeding with the diets containing cimetidine (Hrh2 antagonist) and clobenpropit (Hrh3 antagonist/inverse agonist) significantly lowered the multiplicity of colonic adenocarcinoma. Terfenadine (Hrh1 antagonist) did not affect AOM-DSS-induced colorectal carcinogenesis. Adenocarcinoma cells immunohistochemically expressed Hrh1, Hrh2, Hrh3 and Hrh4 with varied intensities. Because clobenpropit is also known to be a Hrh4 receptor agonist, Hrh2, Hrh3 and Hrh4 may be involved in inflammation-related colorectal carcinogenesis. Additional data, including the mRNA expression of pro-inflammatory cytokines and inducible inflammatory enzymes in the colonic mucosa, are also presented.

Keywords: antagonists; azoxymethane; chemoprevention; colorectal cancer; dextran sodium sulfate; histamine receptors; inflammatory bowel disease.

Figure 1

Representative images of proliferation colorectal lesions induced by AOM and DSS. (A) high grade dysplasia; (B) tubular adenoma; (C) adenocarcinoma without invasion; and (D) adenocarcinoma with invasion. H & E stain, 400×.

Figure 2

(A) Proliferating activities estimated by the MCM2-positive rate of cancer cells in Groups 1 through 4. Insert: MCM2 immunohistochemistry of adenocarcinoma developed in a mouse belonging to Group 1; (B) Apoptosis estimated by the caspase 3-positive rate of cancer cells in Groups 1 through 4. Insert: Caspase 3 immunohistochemistry of adenocarcinoma developed in a mouse belonging to Group 1.

Figure 2

(A) Proliferating activities estimated by the MCM2-positive rate of cancer cells in Groups 1 through 4. Insert: MCM2 immunohistochemistry of adenocarcinoma developed in a mouse belonging to Group 1; (B) Apoptosis estimated by the caspase 3-positive rate of cancer cells in Groups 1 through 4. Insert: Caspase 3 immunohistochemistry of adenocarcinoma developed in a mouse belonging to Group 1.

Figure 3

(A) Immunohistochemical expression of Hrh1, Hrh2, Hrh3 and Hrh4 in normal crypts. H & E stain, 400×; (B) Immunohistochemistry of Hrh1, Hrh2, Hrh3 and Hrh4 in adenocarcinomas developed in the mice of Group 1 (AOM + DSS) and (C) Groups 2 (AOM + DSS + terfenadine), 3 (AOM + DSS + cimetidine) and 4 (AOM + DSS + clobenpropit). Immunohistochemistry, 400×.

Figure 3

(A) Immunohistochemical expression of Hrh1, Hrh2, Hrh3 and Hrh4 in normal crypts. H & E stain, 400×; (B) Immunohistochemistry of Hrh1, Hrh2, Hrh3 and Hrh4 in adenocarcinomas developed in the mice of Group 1 (AOM + DSS) and (C) Groups 2 (AOM + DSS + terfenadine), 3 (AOM + DSS + cimetidine) and 4 (AOM + DSS + clobenpropit). Immunohistochemistry, 400×.

Figure 4

The mRNA expression levels of inducible inflammation-related enzymes and pro-inflammatory cytokines in colorectal tissue samples from mice treated with DSS with or without Hrh antagonists. Total RNA was extracted from the colorectal tissues of untreated and DSS/antagonists-treated mice. The mRNA levels of (A) COX-2, (B) iNOS, (C) TNF-α, (D) IL-1β and (E) IL-6 were measured by quantitative RT-PCR. Treatment with cimetidine or clobenpropit in the diet significantly decreased the mRNA expression of all molecules (* p < 0.05). The expression was normalized to the β-actin mRNA expression level. The data are expressed as the means ± SD from three independent assays (n=8 from each group). The ordinates show the relative mRNA expression (/β-actin).