Galectin-3 as a potential therapeutic target in tumors arising from malignant endothelia

Abstract

Angiosarcoma (ASA) in humans and hemangiosarcoma (HSA) in dogs are deadly neoplastic diseases characterized by an aggressive growth of malignant cells with endothelial phenotype, widespread metastasis, and poor response to chemotherapy. Galectin-3 (Gal-3), a beta-galactoside-binding lectin implicated in tumor progression and metastasis, endothelial cell biology and angiogenesis, and regulation of apoptosis and neoplastic cell response to cytotoxic drugs, has not been studied before in tumors arising from malignant endothelia. Here, we tested the hypothesis that Gal-3 could be widely expressed in human ASA and canine HSA and could play an important role in malignant endothelial cell biology. Immunohistochemical analysis demonstrated that 100% of the human ASA (10 of 10) and canine HSA (17 of 17) samples analyzed expressed Gal-3. Two carbohydrate-based Gal-3 inhibitors, modified citrus pectin (MCP) and lactulosyl-l-leucine (LL), caused a dose-dependent reduction of SVR murine ASA cell clonogenic survival through the inhibition of Gal-3 antiapoptotic function. Furthermore, both MCP and LL sensitized SVR cells to the cytotoxic drug doxorubicin to a degree sufficient to reduce the in vitro IC(50) of doxorubicin by 10.7-fold and 3.6-fold, respectively. These results highlight the important role of Gal-3 in the biology of ASA and identify Gal-3 as a potential therapeutic target in tumors arising from malignant endothelial cells.

Keywords: Angiosarcoma; apoptosis; chemotherapy; doxorubicin; galectin-3.

Figures

Figure 1

Immunohistochemical analysis of Gal-3 expression using TIB-166 rat anti-Gal-3 monoclonal antibody in human ASA (A and B) and canine HSA (C and D). Brown staining in (A) and (C) represents Gal-3 immunoreactivity. (B) and (D) show corresponding negative controls omitting a primary antibody. (E–H) An example of the computer-assisted analysis of images shown in (C) and (D) using ImageProPlus software. The results of a computer-assisted analysis correlated well with the scores made by the observers in samples with high and moderate Gal-3 expressions. However, in samples with negative or weak Gal-3 expression, computer-assisted analysis yielded often elevated (false-positive) scores. Slides were counterstained with hematoxylin. Scale bars, 100 µm.

Figure 2

Differential diagnosis between Gal-3 and hemosiderin staining in canine splenic HSA samples. In (A)–(D), an HSA sample was characterized using H&E staining (A), anti-Gal-3 antibody (B), nonimmune control (C), and anti-von Willebrand factor polyclonal antibody (D). Brown staining in (B) indicates Gal-3 immunoreactivity. Brown staining in (D) shows von Willebrand factor immunoreactivity consistent with the endothelial origin of HSA cells. In (E)–(H), a sample of normal canine spleen tissue is shown. Note the presence of brown staining material in H&E slides (E, black arrows) and nonimmune control (G, red arrows) identified as hemosiderin deposits using Prussian blue stain for iron (H, green arrows). Scale bar shown in (H), 100 µm.

Figure 3

Immunohistochemical analysis (A) and Western blot analysis (B) confirmation of Gal-3 expression in the murine ASA cell line SVR. In (A), brown staining indicates Gal-3 immunoreactivity. Note the predominantly cytoplasmic Gal-3 localization (black arrow) with limited nuclear positivity (red arrow). In (B), a single Gal-3 immunoreactive band was identified by Western blot analysis.

Figure 4

The effect of the small-molecular-weight carbohydrate-based Gal-3 inhibitors MCP and LL on the clonogenic survival of SVR cells. In (A)–(C), SVR cells were plated at low density (200 cell/well) in 24-well plates in increasing concentrations of MCP (0–0.5%) and LL (0–1 mM). Seven days later, colonies of ≥ 15 cells were scored. Both MCP (A and C) and LL (B) inhibited the clonogenic survival of SVR cells in a dose-dependent manner. TUNEL analysis (D–G) demonstrated that the effect of both MCP (D and F) and LL (D and G) on SVR clonogenic survival was associated with a significant reduction in the percentage of TUNEL-negative (nonapoptotic) cells in samples treated with MCP (F) and LL (G) compared to untreated control (E). Note the presence of nonapoptotic cells in the untreated control (E, black arrows) versus an almost complete absence of TUNEL-negative cells in samples treated with MCP (F) or LL (G).

Figure 5

The effect of the carbohydrate-based Gal-3 inhibitors MCP and LL on SVR cell sensitivity to doxorubicin. Both MCP (A) and LL (B) sensitize SVR cells to doxorubicin. Note the significant shift to the left of graphs representing a combined effect of doxorubicin with the ICmin of MCP (A, open circles) and LL (B, open circles) compared to the effect of doxorubicin alone (A and B, closed circles), or a would-be-additive-effect graph (A and B, red line) on the clonogenic survival of SVR. (C) In vitro IC50of doxorubicin alone or in combination with the ICmin of MCP (0.06%) or LL (200 µM).