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Galectin-9 (Gal-9) induced the apoptosis of not only T cell lines but also of other types of cell lines in a dose- and time-dependent manner. The apoptosis was suppressed by lactose, but not by sucrose, indicating that β-galactoside binding is essential for Gal-9-induced apoptosis. Moreover, Gal-9 required at least 60 min of Gal-9 binding and possibly de novo protein synthesis to mediate the apoptosis. We also assessed the apoptosis of peripheral blood T cells by Gal-9. Apoptosis was induced in both activated CD4+ and CD8+ T cells, but the former were more susceptible than the latter. A pan-caspase inhibitor (Z-VAD-FMK) inhibited Gal-9-induced apoptosis. Furthermore, a caspase-1 inhibitor (Z-YVAD-FMK), but not others such as Z-IETD-FMK (caspase-8 inhibitor), Z-LEHD-FMK (caspase-9 inhibitor), and Z-AEVD-FMK (caspase-10 inhibitor), inhibited Gal-9-induced apoptosis. We also found that a calpain inhibitor (Z-LLY-FMK) suppresses Gal-9-induced apoptosis, that Gal-9 induces calcium (Ca2+) influx, and that either the intracellular Ca2+ chelator BAPTA-AM or an inositol trisphosphate inhibitor 2-aminoethoxydiphenyl borate inhibits Gal-9-induced apoptosis. These results suggest that Gal-9 induces apoptosis via the Ca2+-calpain-caspase-1 pathway, and that Gal-9 plays a role in immunomodulation of T cell-mediated immune responses.

A 1.6-kilobase pair cDNA was isolated from a human T-cell-derived expression library that encodes a novel eosinophil chemoattractant (designated ecalectin) expressed during allergic and parasitic responses. Based on its deduced amino acid sequence, ecalectin is a 36-kDa protein consisting of 323 amino acids. Although ecalectin lacks a hydrophobic signal peptide, it is secreted from mammalian cells. Ecalectin is not related to any known cytokine or chemokine but rather is a variant of human galectin-9, a member of the large family of animal lectins that have affinity for ␤-galactosides. Recombinant ecalectin, expressed in COS cells and insect cells, exhibited potent eosinophil chemoattractant activity and attracted eosinophils in vitro and in vivo in a dose-dependent manner but not neutrophils, lymphocytes, or monocytes. The finding that the ecalectin transcript is present in abundance in various lymphatic tissues and that its expression increases substantially in antigen-activated peripheral blood mononuclear cells suggests that ecalectin is an important T-cell-derived regulator of eosinophil recruitment in tissues during inflammatory reactions. We believe that this is the first report of the expression of an immunoregulatory galectin expressed by a T-cell line that is selective for eosinophils.

Galectin-9 expression was examined in 6 human melanoma cell lines. Among them, MM-BP proliferated with colony formation, but MM-RU failed. RT-PCR analysis revealed evident expression of galectin-9 mRNA in MM-BP but not in MM-RU. MM-BP expressed galectin-9 protein both on the surface and in the cytoplasm, whereas MM-RU expressed it only weakly in the cytoplasm. Exogenous galectin-9 induced in vitro both cell aggregation and apoptosis of MM-RU proliferating without colony formation. Association of galectin-9 expression in melanoma cells with prognosis of the patients bearing melanocytic tumors was further examined. Galectin-9 protein was strongly and homogeneously expressed in melanocytic nevi, but down-regulated in melanoma cells especially in metastatic lesions. High galectin-9 expression was inversely correlated with the progression of this disease, suggesting that high galectin-9 expression in primary melanoma lesions links to a better prognosis.

Purpose: Galectin-9, a member of the h-galactoside^binding galectin family, induces aggregation of certain cell types.We assessed the contribution of galectin-9 to the aggregation of breast cancer cells as well as the relation between galectin-9 expression in tumor tissue and distant metastasis in patients with breast cancer. Experimental Design: Subclones of MCF-7 breast cancer cells with high or low levels of galectin-9 expression were established and either cultured on plastic dishes or transplanted into nude mice. The tumors of 84 patients with breast cancer were tested for galectin-9 expression by immunohistochemistry. The patients were followed up for 14 years. Results: MCF-7 subclones with a high level of galectin-9 expression formed tight clusters during proliferation in vitro, whereas a subclone (K10) with the lowest level of galectin-9 expression did not. However, K10 cells stably transfected with a galectin-9 expression vector aggregated in culture and in nude mice. Ectopic expression of galectin-9 also reduced MCF-7 cell adhesion to extracellular matrix proteins.Tumors of 42 of the 84 patients were galectin-9 positive, and those of 19 of the 21patients with distant metastasis were galectin-9 negative. None of the13 patients with galectin-9^positive tumors and lymph node metastasis up to level II manifested distant metastasis.The cumulative disease-free survival ratio for galectin-9^positive patients was more favorable than that for the galectin-9^negative group (P < 0.0001). Multivariate analysis revealed that galectin-9 status influenced distant metastasis independently of and to a greater extent than lymph node metastasis. Conclusions: Galectin-9 is a possible prognostic factor with antimetastatic potential in breast cancer.Distant metastasis is a major clinical determinant of the survival of individuals with breast cancer. The number of lymph node metastases (node status) has long been used to predict distant metastasis in breast cancer. The various biological markers proposed for the prediction of distant metastasis in breast cancer include loss of nm23 expression (1, 2), with increased expression of this gene induced by inhibition of DNA methylation also having been found to prevent distant metastasis (3). The levels of both total and low molecular weight cyclin E, as determined by immunoblot analysis, are also correlated with survival in patients with breast cancer, especially in those with node-negative cancer (4).However, none of these biological markers is as effective as node status in the prediction of distant metastasis or is suitable as an indicator of the need for adjuvant chemotherapy.Galectin-9 is a member of the h-galactoside-binding galectin family of proteins (5-8). Among the members of the galectin family, galectin-1 (9) and galectin-3 (10 -13) contribute to tissue invasion by and metastasis of several types of cancer cells, including breast cancer cells. Galectin-3 also serves as a marker for preoperative diagnosis of nodular thyroid lesions (14). We have recently shown ...