Abstract
In order to study the role of galectin-3 in tumor angiogenesis associated with tumor-associated macrophages (TAM) and tumor parenchyma, the galectin-3 expression was reconstituted in Tm1 melanoma cell line that lacks this protein. Galectin-3-expressing cells (Tm1G3) and mock-vector transfected cells (Tm1N3) were injected into wild-type (WT) and galectin-3 knockout (KO) C57Bl/6 mice. Tumors originated from Tm1G3 were larger in tumor volume with enlarged functional vessels, decreased necrotic areas, and increased vascular endothelial growth factor (VEGF) protein levels. Galectin-3-nonexpressing-cells injected into WT and KO showed increased levels of transforming growth factor beta 1 (TGFβ1) and, in WT animals this feature was also accompanied by increased VEGFR2 expression and its phosphorylation. In KO animals, tumors derived from galectin-3-expressing cells were infiltrated by CD68+-cells, whereas in tumors derived from galectin-3-nonexpressing-cells, CD68+ cells failed to infiltrate tumors and accumulated in the periphery of the tumor mass. In vitro studies showed that Tm1G3 secreted more VEGF than Tm1N3 cells. In the latter case, TGFβ1 induced VEGF production. Basal secretion of VEGF was higher in WT-bone marrow-derived macrophages (BMDM) than in KO-BMDM. TGFβ1 induced secretion of VEGF only in WT-BMDM. Tm1G3-induced tumors had the Arginase I mRNA increased, which upregulated alternative macrophage (M2)/TAM induction. M2 stimuli, such as interleukin-4 (IL4) and TGFβ1, increased Arginase I protein levels and galectin-3 expression in WT- BMDM, but not in cells from KO mice. Hence, we report that galectin-3 disruption in tumor stroma and parenchyma decreases angiogenesis through interfering with the responses of macrophages to the interdependent VEGF and TGFβ1 signaling pathways.
Highlights
Galectin-3, a conserved b-galactoside-binding animal lectin, has important physiological roles, such as development and activation of the immune system, besides its involvement in tumor pathophysiology [1]
We evaluated the protein levels of galectin-3 in Tm1 cells, as compared to Melan-A and observed a total absence of this lectin in Tm1 cells, suggesting that galectin-3 expression was downregulated upon malignant transformation
It is well known that aberrant methylation of CpG dinucleotides is responsible for gene silencing [28] and, based on that, we first performed in silico analyses to identify putative CpG islands in the promoter region of the galectin-3 murine gene (GenBank sequence number L08649.1)
Summary
Galectin-3, a conserved b-galactoside-binding animal lectin, has important physiological roles, such as development and activation of the immune system, besides its involvement in tumor pathophysiology [1]. In experiments with Human Umbilical Vein Endothelial Cells (HUV-EC-C), Nangia-Makker et al [3] described that neutralizing galectin-3 by specific carbohydrates and anti-galectin-3 antibodies affect chemotaxis, endothelial cell morphology, and capillary tube formation in vitro. These authors demonstrated that galectin-3 has pro-angiogenic activity, which may be related to its ability to induce endothelial cell migration. Markowska et al [4] showed in vitro that galectin-3 siRNA knockdown as well as galectin-3 blockers resulted in reduction in angiogenesis induced by VEGF and basic fibroblast growth factor (bFGF) These authors demonstrated that VEGF and bFGF-induced angiogenesis in mouse corneal micropocket assay was reduced in galectin-3 knockout (KO) mice. They provided evidence to support the notion that galectin-3 generates VEGFR2clusters, potentiating cell signaling effects in endothelial cells [5]
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