A novel clinically relevant animal model for studying galectin-3 and its ligands during colon carcinogenesis

Abstract

Galectin-3 (Gal-3) is a multifunctional protein that plays different roles in cancer biology. To better understand the role of Gal-3 and its ligands during colon carcinogenesis, we studied its expression in tumors induced in rats treated with 1,2-dimethylhydrazine (DMH) and in human tissues. Normal colon from untreated rats showed no staining using two specific monoclonal antibodies. In contrast, morphologically normal colon from DMH-treated rats and dysplastic aberrant crypt foci were strongly stained, indicating that increased Gal-3 expression is an early event during the neoplastic transformation in colon cells. Gal-3 was weakly expressed in adenocarcinomas. Overall, the Gal-3 expression pattern observed in the DMH rat model closely resembles that displayed by human colon stained with the same antibodies. We also found that Gal-3 phosphorylation diminishes in serines while increasing in tyrosines during rat colon carcinogenesis. Finally, we showed that Gal-3-ligands expression is strikingly similar in rat and human malignant colon and in non-malignant tissues. In conclusion, the DMH-induced rat colon cancer model displays expression patterns of Gal-3 and its ligands very similar to those observed in human samples. This animal model should contribute to clarifying the role of Gal-3 in colon carcinogenesis and also to finding effective preventive cancer agents based on Gal-3 targeting.

Figure 1

Galectin-3 (Gal-3) expression during rat colon carcinogenesis. Anti–Gal-3 monoclonal antibodies (MAbs) A3A12 and A1D6 were probed against colonic rat tissues. Positive cells are shown in brown (DAB staining) and counter-colorated with hematoxylin (blue staining). Images are representative of different animals for each condition. (A,G) Normal colon from untreated rats. (B,C,H,I) Morphologically normal colon from 1,2-dimethylhydrazine (DMH)-treated rats at 32 weeks of carcinogenesis. (D,E,J,K) DMH-induced aberrant crypt foci at 40 weeks of carcinogenesis. (F,L) Hepatic metastasis from DMH-induced colon adenocarcinoma at 40 weeks of carcinogenesis.

Figure 2

Semi-quantification of A3A12 and A1D6 staining during rat colon carcinogenesis. Semi-quantification was performed by two independent investigators (DM and MR). After immunoperoxidase staining with anti–Gal-3 MAbs, tissue sections were evaluated. Six different animals were studied in each group.

Figure 3

Gal-3 expression in human colonic tissues. Anti–Gal-3 MAbs A3A12 and A1D6 were probed against colonic human tissues. Positive cells are shown in brown (DAB staining) and counter-colorated with hematoxylin (blue staining). Images are representative of samples from twelve different patients. (A,F) Morphologically normal colon mucosa distant from the tumor. (B,G) Morphologically normal colon mucosa adjacent to the tumor. (C,D,H,I,E,J) Colon adenocarcinoma. E and J are magnifications of the areas in the squares shown in D and I, respectively.

Figure 4

Semi-quantification of A3A12 and A1D6 staining in human colon adenocarcinomas. Each number on the x-axis represents one patient. (A) Well-differentiated tumors. (B) Moderately differentiated tumors. (C) Poorly differentiated tumors. Semi-quantification was performed by two independent investigators as described in Materials and Methods.

Figure 5

A3A12-negative human colon adenocarcinomas express Gal-3. (A) RT-PCR showing Gal-3 and β-2 microglobulin (internal control) mRNA expression in colon adenocarcinoma samples from the indicated patients (numbers in the upper part of the image). (B) Immunohistochemical staining of the same tumors using the anti–Gal-3 MAb TIB-166. Immunostaining is shown in brown, whereas blue staining represents hematoxylin counter-coloration.

Figure 6

Gal-3 phosphorylation and expression pattern during rat colon carcinogenesis. Cell lysates from untreated rats, normal mucosa from DMH-treated rats, and colon carcinomas were immunoprecipitated using the anti–Gal-3 MAb TIB-166. After that, Western blot analyses of phosphoserine, phosphotyrosine, and Gal-3 were performed with appropriate antibodies. (A) In the upper panel, representative Western blots are depicted. In the digitalized image, the depicted lanes were spliced from the original file. In the lower panel, the mean ± SD densitometric signal is shown for each antibody used. (B) Phosphoserine/Gal-3 and phosphotyrosine/Gal-3 ratios from densitometric analysis of Western blot. Results are plotted as the ratio of phospho-Ser or phosphor-Tyr to Gal-3 ± SD of four independent experiments.

Figure 7

Gal-3 ligand expression during DMH-induced rat colon carcinogenesis and in human malignant and non-malignant colon. Gal-3 ligands were identified by incubating Gal-3–horseradish peroxidase (brown staining) in rat (A–C) or human (D–F) colon tissues. (A) Normal mucosa from untreated rats. (B) Morphologically normal mucosa from DMH-treated rats (32 weeks of carcinogenesis). (C) Rat colon adenocarcinoma. (D) Morphologically normal colon mucosa distant from the tumor. (E) Morphologically normal mucosa surrounding the tumor. (F) Human colon adenocarcinoma. Arrows, caliciform cells; arrowheads, apical surface of epithelial cells. Images are representative of samples from twelve different patients and six different rats for each condition.

Figure 8

Alignment of human and rat Gal-3 amino acids sequence (aa 1–50) depicting the epitopic sequences recognized by A3A12, A1D6, and TIB166 MAbs. Already described (serine) and putative phosphorylable (tyrosine) residues are indicated by arrows.