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. 2010 Apr;31(4):729-36.
doi: 10.1093/carcin/bgq002. Epub 2010 Jan 8.

Tumor Formation in a Mouse Model of Colitis-Associated Colon Cancer Does Not Require COX-1 or COX-2 Expression

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Free PMC article

Tumor Formation in a Mouse Model of Colitis-Associated Colon Cancer Does Not Require COX-1 or COX-2 Expression

Tomo-O Ishikawa et al. Carcinogenesis. .
Free PMC article

Abstract

Cyclooxygenase-2 (COX-2), a key enzyme of prostanoid biosynthesis, plays an important role in both hereditary and spontaneous colon cancer. Individuals with ulcerative colitis are also at high risk for colorectal cancer. To investigate the role of Cox-2 in colitis-associated colon cancer, we subjected Cox-2 luciferase-knock-in mice and Cox-2-knockout mice to a well-known mouse model of colitis-associated cancer in which animals are treated with a single-azoxymethane (AOM) injection followed by dextran sulfate sodium (DSS) administration. Tumors induced by AOM and DSS expressed significantly higher Cox-2 levels when compared with surrounding areas of colon, as detected both by luciferase reporter gene expression driven from the endogenous Cox-2 promoter and by western blotting of COX-2 protein in Cox-2 luciferase heterozygous knock-in mice. Immunofluorescence revealed that tumor stromal fibroblasts, macrophages and endothelial cells express COX-2 protein. In contrast, little COX-2 expression was observed in myofibroblasts or epithelial cells. Despite a significant elevation of COX-2 expression in AOM/DSS-induced colon tumors in wild-type mice, similar tumors developed in AOM/DSS-treated Cox-2(-/-)- and Cox-1(-/-)-knockout mice. These results indicate that cyclooxygenase-derived prostanoids are not major players in colitis-associated cancer. In contrast, tumor formation induced by multiple injections of AOM (with no DSS-induced colitis) did not occur in Cox-2(-/-)-knockout mice. Our data suggest that the mechanism of colorectal tumor promotion in colitis-associated cancer differs from the mechanism of tumor promotion for hereditary and sporadic colorectal cancer.

Figures

Fig. 1.
Fig. 1.
COX-2 and luciferase expression in AOM/DSS-induced Cox-2luc/+ colon tumors. (A and B) Ex vivo imaging of luciferase expression in the colon of a Cox-2luc/+ mouse 12 weeks after AOM injection. Photo (A) shows a fixed opened colon with tumors. The color overlay on the image (B) illustrates the photons per second emitted from tissue, as shown in pseudocolor scales next to the image. (C) Quantification of luciferase activity determined by bioluminescent imaging. A region of interest (ROI) was manually selected on each tumor and each normal area. The area of the ROI was kept constant. Average photon number was measured. (D) Luciferase activity, measured by in vitro luciferase assay of tissue lysates, in tumors and normal colon regions. The same tissues analyzed in (C) were homogenized and lysates were assayed. (E) Western blot analysis of COX-2 protein expression in tumors and normal areas of the colon. Lysates used for luciferase assay in (D) were also used to detect COX-2 protein. GAPDH is a loading control.
Fig. 2.
Fig. 2.
Immunofluorescent detection of COX-2-expressing cells. Dual-color double-staining immunofluorescent analyses of proteins expressed in AOM–DSS-induced colorectal tumors from wild-type mice, performed with anti-COX-2 and antibody to cell type-specific marker proteins; (A) anti-F4/80 for macrophage, (B) anti-vimentin for fibroblasts, (C) anti-α-smooth muscle actin for myofibroblasts, (D) pan-keratin antibody for epithelial cells and (E) anti-CD34 for endothelial cells. For each row, the two antibodies used for immunofluorescence are named in the right panel; the color of the antibody staining is indicated by the color of the font.
Fig. 3.
Fig. 3.
AOM/DSS-induced colon tumor formation in Cox-2/-knockout mice. (A) Viability of mice following AOM and DSS administration. (B) Numbers of tumors per mouse in Cox-2/-knockout and Cox-2+/+ wild-type mice. Vertical bars indicate the mean tumor number and SE. (C) Histological sections of tumors in wild-type Cox-2+/+ (left panels) and Cox-2/-knockout (right panels) mice.
Fig. 4.
Fig. 4.
AOM/DSS-induced colon tumor formation in Cox-2luc/-knockout mice. (A) Viability of mice following AOM and DSS administration. (B) Numbers of tumors per mouse in Cox-2luc/-knockout and Cox2+/+ wild-type mice. Vertical bars indicate the mean tumor number and SE. (C) Histological sections of tumors in wild-type Cox-2+/+ (left panels) and Cox-2luc/-knockout (right panels) mice.
Fig. 5.
Fig. 5.
AOM/DSS-induced colon tumor formation in Cox-1/-knockout mice. (A) Body weight of mice following AOM and DSS administration. (B) Numbers of tumors per mouse in Cox-1/-knockout and Cox1+/+ wild-type mice. Vertical bars indicate the mean tumor number and SE. (C) Histological sections of tumors in wild-type Cox-1+/+ (left panels) and Cox-1/-knockout (right panels) mice.
Fig. 6.
Fig. 6.
Colon tumor formation in Cox-2-knockout mice in response to repeated AOM injection. (A) Immunohistochemical detection of COX-2 expression in a tumor induced by repeated AOM injection. (B) Numbers of tumors per mouse in Cox-2/-knockout and Cox-2+/+ control mice. Vertical bars indicate the mean polyp number and SE.

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