Tumor-associated antigen 90K/Mac-2-binding protein: possible role in colon cancer

The tumor-associated antigen 90K (TAA90K)/Mac-2-binding protein implicated in cancer progression and metastasis is modified by beta1-6 branched N-linked oligosaccharides in colon cancer cells, glycans shown to contribute to cancer metastasis. To elucidate the role of TAA90K in colon cancer, we examined its expression and function in human colon tumors and colon carcinoma cell lines. Immunohistochemical analyses of colon tumors revealed elevated expression of TAA90K in all samples analyzed compared to normal colon. To examine the function of TAA90K in colon cancer, we carried out protein and cell binding assays using TAA90K-His purified from HT-29 cells colon carcinoma cells infected with recombinant vaccinia virus expressing TAA90K containing a C-terminal poly-histidine tag. TAA90K-His bound to fibronectin, collagen IV, laminins-1, -5, and -10 and galectin-3 (Mac-2) but poorly to collagen I and galectin-1. As expected, binding of TAA90K to galectin-3 was dependent on carbohydrate since it was inhibitable by lactose and asiolofetuin, and a TAA90K-His glycoform purified from HT-29 cells treated with the glycosylation inhibitor 1-deoxymannojirimycin bound poorly to galectin-3. Unlike TAA90K isolated from other cell types, TAA90K-His isolated from colon cancer cells failed to mediate adhesion of colon cancer and normal cell lines, possibly due to cell-type specific glycosylation of TAA90K-His and/or its putative cellular receptor. However, at low concentrations, TAA90K-His enhanced galectin-3-mediated HT-29 cell adhesion while at high concentrations, it inhibited cell adhesion. Thus, a possible mechanism by which TAA90K may contribute to colon cancer progression is by modulating tumor cell adhesion to extracellular proteins, including galectin-3.     

半乳糖凝集素-3(galectin-3)基因沉默对Eca109人类食管癌细胞的影响

半乳糖凝集素-3(galectin-3)是一种多功能的β-半乳糖苷结合凝集素,涉及包括细胞生长、粘附、增殖、进展、转移以及凋亡等多种生物学功能,在恶性肿瘤中高表达。以前的研究已经证实了galecin-3过表达在Eca109人食管癌细胞的生物学作用。本研究试图通过进行小干扰RNA(siRNA)介导的galectin-3沉默,以分析galectin-3沉默对食管癌细胞生物学行为的影响。我们采用Western blotting和RT-qPCR被用来证实在蛋白质和mRNA水平上的galectin-3低表达,使用细胞计数试剂盒-8评估细胞增殖,用Annexin V-PE/7-AAD细胞凋亡检测试剂盒和流式细胞术检测Eca109细胞的凋亡。研究结果表明,转染后72 h,si Gal-3组Eca109细胞增殖明显低于siRNA对照组和未处理组(p<0.001)。Transwell实验结果显示,与其他组相比,galecin-3的抑制作用显著降低Eca109细胞的迁移和侵袭能力(p<0.05)。与siRNA-对照组和未处理组相比,galectin-3敲低显著增加Eca109细胞的凋亡率(p<0.05)。敲低Eca109细胞中galecin-3的表达后,细胞增殖、迁移和侵袭能力下降,而细胞凋亡增强,说明galectin沉默可作为食管癌治疗的新策略。     

Multifaceted role of galectin-3 on human glioblastoma cell motility

Astrocytic tumors' aggressiveness results from an imbalance between cell proliferation and cell death favoring growth, but also from the propensity of tumor cells to detach from the primary tumor site, migrate, and invade the surrounding parenchyma. Astrocytic tumor progression is known to be associated with an increased expression of galectin-3. We investigated in cell culture how galectin-3 expression affects astrocytoma cell motility. Galectin-3 deficient cells were obtained by stable transfection of the U373 glioblastoma cell line with a specific expression antisense plasmid. Cultured galectin-3 deficient glioblastoma cells showed increased motility potential on laminin and modifications in the cytoskeleton reorganization. In addition, c-DNA microarrays and quantitative immunofluorescence analysis showed that galectin-3 deficient U373 cells have an increased expression of integrins-alpha6 and -beta1, proteins known to be implicated in the regulation of cell adhesion.     

Galectin-1 knocking down in human U87 glioblastoma cells alters their gene expression pattern

We have previously reported that (i) progression of malignancy in patients bearing astrocytic tumors correlates with increased tumor levels of galectin-1; (ii) in vitro addition of purified galectin-1 to U87 human glioblastoma cells enhances tumor cell motility; and (iii) conversely, knocking down galectin-1 expression in this cell line by stable transfection with antisense galectin-1 mRNA impairs motility and delays mortality after their intracranial grafting to nude mice. We here used cDNA microarray analysis to compare the effect on gene expression of stable transfection with antisense galectin-1 vector to mock-transfected and wild-type cells. Among the 631 spots probing genes potentially involved in cancer that were valid for analysis on all the arrays the expression of 86 genes was increased at least 2-fold. Confirmation of increased protein levels was provided by immunocytochemistry for p21waf/cip1, cullin-2, p53, ADAM-15, and MAP-2. Major differences in the expression patterns of ADAM-15 and the actin stress fiber organization were also observed. U87 cells stably deficient for galectin-1 expression were significantly less motile than control. We conclude that the stable inhibition of galectin-1 expression alters the expression of a number of genes that either directly or indirectly influence adhesion, motility and invasion of human glioblastoma cells.     

Colon cancer cells secreted CXCL11 via RBP-Jκ to facilitated tumour-associated macrophage-induced cancer metastasis

Metastasis is the main cause of colon cancer-related deaths. RBP-Jκ is involved in colon cancer development, but its function in colon cancer metastasis is still unclear. Tumour-associated macrophages are the main cell components in tumour microenvironments. Here, we aimed to determine the function of RBP-Jκ in colon cancer metastasis and its underlying mechanisms for modulating interactions between colon cancer cell and tumour-associated macrophages. Through bioinformation analysis, we found that RBP-Jκ was overexpressed in colon cancer tissues and associated with advanced colon cancer phenotypes, macrophage infiltration and shorter survival overall as confirmed by our patients’ data. And our patients’ data show that RBP-Jκ expression and tumour-associated macrophages infiltration are associated with colon cancer metastasis and are independent prognostic factors for colon cancer patients. Tumour-associated macrophages induced colon cancer cell migration, invasion and epithelial-mesenchymal transition through secreting TGF-β1. Colon cancer cells with high RBP-Jκ expression induced the expression of TGF-β1 in tumour-associated macrophages by secreting CXCL11. Our research revealed that colon cancer cells secreted CXCL11 via overexpression of RBP-Jκ to enhance the expression of TGF-β1 in tumour-associated macrophages to further promote metastasis of colon cancer cells.     

Regulation of melanoma metastasis to lungs by cell surface Lysosome Associated Membrane Protein-1 (LAMP1) via galectin-3

Lysosome Associated Membrane Protein-1 (LAMP1), which lines the lysosomes, is often found to be expressed on surface of metastatic cells. We previously demonstrated that its surface expression on B16 melanoma variants correlates with metastatic potential. To establish the role of cell surface LAMP1 in metastasis and to understand the possible mechanism by which it facilitates lung colonization, LAMP1 was downregulated in high metastatic B16F10 cells using shRNAs cloned in a doxycycline inducible vector. This also resulted in significantly decreased LAMP1 on the cell surface. Being a major carrier of poly-N-acetyllactosamine (polyLacNAc) substituted β1,6 branched N-oligosaccharides, the high affinity ligands for galectin-3, LAMP1 down regulation also resulted in appreciably decreased binding of galectin-3 to the cell surface. LAMP1 has been shown to bind to Extracellular Matrix (ECM), Basement Membrane (BM) components and also to galectin-3 (via carbohydrates) which is known to get incorporated into the ECM and BM. Although, LAMP1 downregulation had a marginal effect on cellular spreading and motility on fibronectin and matrigel, it significantly altered the same on galectin-3, and ultimately leading to notably reduced lung metastasis. The results thus for the first time provide direct evidence that cell surface LAMP1 facilitates lung metastasis by providing ligands for galectin-3 which has been shown to be expressed in highest amounts on lungs and constitutively on its vascular endothelium.     

Mechano-transduction mediated secretion and uptake of galectin-3 in breast carcinoma cells: implications in the extracellular functions of the lectin

In the following experiments, we sought to understand the triggering mechanism which propels galectin-3 to be secreted into the extracellular compartment from its intracellular stores in breast carcinoma cells. We also wanted to analyze in greater details the role of galectin-3 in cellular adhesion and spreading. To do this, we made use of two pairs of breast carcinoma cell lines where one of the pair has high expression of galectin-3 and the other low expression of the lectin. We determined that galectin-3 secreted into the conditioned medium of sub-confluent and spread cells in culture was quite low, almost negligible. However, once the cells were detached and rounded up, a mechano-sensing mechanism triggered the rapid secretion of galectin-3 into the conditioned medium. The secretion was constitutive as long as the cells remained detached. Galectin-3 was shown to be actively taken up from the conditioned medium by spreading cells. The cells which express and secrete high levels of galectin-3 adhered and spread much faster on plastic than those with reduced expression. The uptake of galectin-3 according to our data was important in cell spreading because if this process was compromised significantly, cells failed to spread. The data suggested that galectin-3 uptake modulates the adhesion plaques in that cells which express high levels of galectin-3 have thin-dot like plaques that may be suited for rapid adhesion and spreading while cells in which galectin-3 expression is reduced or knocked-down, have thick and elongated plaques which may be suited for a firmer adhesion to the substratum. Recombinant galectin-3 added exogenously reduced the thickness of the adhesion plaques of tumor cells with reduced galectin-3 expression. Taken together, the present data suggest that galectin-3 once externalized, is a powerful modulator of cellular adhesion and spreading in breast carcinoma cells.     

L1, a novel target of beta-catenin signaling, transforms cells and is expressed at the invasive front of colon cancers

Aberrant beta-catenin-TCF target gene activation plays a key role in colorectal cancer, both in the initiation stage and during invasion and metastasis. We identified the neuronal cell adhesion molecule L1, as a target gene of beta-catenin-TCF signaling in colorectal cancer cells. L1 expression was high in sparse cultures and coregulated with ADAM10, a metalloprotease involved in cleaving and shedding L1's extracellular domain. L1 expression conferred increased cell motility, growth in low serum, transformation and tumorigenesis, whereas its suppression in colon cancer cells decreased motility. L1 was exclusively localized in the invasive front of human colorectal tumors together with ADAM10. The transmembrane localization and shedding of L1 by metalloproteases could be useful for detection and as target for colon cancer therapy.     

Phosphorylation of the beta-galactoside-binding protein galectin-3 modulates binding to its ligands

The beta-galactoside-binding protein galectin-3 has pleiotropic biological functions and has been implicated in cell growth, differentiation, adhesion, RNA processing, apoptosis, and malignant transformation. Galectin-3 may be phosphorylated at N-terminal Ser(6), but the role of phosphorylation in determining interactions of this endogenous lectin with its ligands remains to be elucidated. We therefore studied the effect of phosphorylation on binding of galectin-3 to two of its reported ligands, laminin and purified colon cancer mucin. Human recombinant galectin-3 was phosphorylated in vitro by casein kinase I, and separated from the native species by isoelectric focusing for use in solid phase binding assays. Non-phosphorylated galectin-3 bound to laminin and asialomucin in a dose-dependent manner with half-maximal binding at 1.5 microg/ml. Phosphorylation reduced saturation binding to each ligand by >85%. Ligand binding could be fully restored by dephosphorylation with protein phosphatase type 1. Mutation of galectin-3 at Ser(6) (Ser to Glu) did not alter galectin ligand binding. Metabolic labeling or separation by isoelectric focusing confirmed the presence of phosphorylated galectin-3 species in vivo in the cytosol of human colon cancer cells from which ligand mucin was purified. Phosphorylation significantly reduces the interaction of galectin-3 with its ligands. The process by which phosphorylation modulates protein-carbohydrate interactions has important implications for understanding the biological functions of this protein, and may serve as an "on/off" switch for its sugar binding capabilities.     

Galectin-3 protein modulates cell surface expression and activation of vascular endothelial growth factor receptor 2 in human endothelial cells

Angiogenesis is heavily influenced by VEGF-A and its family of receptors, particularly VEGF receptor 2 (VEGF-R2). Like most cell surface proteins, VEGF-R2 is glycosylated, although the function of VEGF-R2 with respect to its glycosylation pattern is poorly characterized. Galectin-3, a glycan binding protein, interacts with the EGF and TGFβ receptors, retaining them on the plasma membrane and altering their signal transduction. Because VEGF-R2 is glycosylated and both galectin-3 and VEGF-R2 are involved with angiogenesis, we hypothesized that galectin-3 binds VEGF-R2 and modulates its signal transduction as well. Employing a Western blot analysis approach, we found that galectin-3 induces phosphorylation of VEGF-R2 in endothelial cells. Knockdown of galectin-3 and Mgat5, an enzyme that synthesizes high-affinity glycan ligands of galectin-3, reduced VEGF-A mediated angiogenesis in vitro. A direct interaction on the plasma membrane was detected between galectin-3 and VEGF-R2, and this interaction was dependent on the expression of Mgat5. Using immunofluorescence and cell surface labeling, we found an increase in the level of internalized VEGF-R2 in both Mgat5 and galectin-3 knockdown cells, suggesting that galectin-3 retains the receptor on the plasma membrane. Finally, we observed reduced suture-induced neovascularization in the corneas of Gal3(-/-) and Mgat5(-/-) mice. These findings are consistent with the hypothesis that, like its role with the EGF and TGFβ receptors, galectin-3 contributes to the plasma membrane retention and proangiogenic function of VEGF-R2.     

Galectin-3 Guides Intracellular Trafficking of Some Human Serotransferrin Glycoforms

Transferrin internalization via clathrin-mediated endocytosis and subsequent recycling after iron delivery has been extensively studied. Here we demonstrate a previously unrecognized parameter regulating this recycling, the binding of galectin-3 to particular glycoforms of transferrin. Two fractions of transferrin, separated by affinity chromatography based on their binding or not to galectin-3, are targeted to kinetically different endocytic pathways in HFL-1 cells expressing galectin-3 but not in SKBR3 cells lacking galectin-3; the SKBR3 cells, however, can acquire the ability to target these transferrin glycoforms differently after preloading with exogenously added galectin-3. In all, this study provides the first evidence of a functional role for transferrin glycans, in intracellular trafficking after uptake. Moreover, the galectin-3-bound glycoform increased in cancer, suggesting a pathophysiological regulation. These are novel aspects of transferrin cell biology, which has previously considered only a degree of iron loading, but not other forms of heterogeneity.     

Galectin binding to Mgat5-modified N-glycans regulates fibronectin matrix remodeling in tumor cells

Oncogenic signaling stimulates the dynamic remodeling of actin microfilaments and substrate adhesions, essential for cell spreading and motility. Transformation is associated with increased expression of beta1,6GlcNAc-branched N-glycans, products of Golgi beta1,6-acetylglucosaminyltransferase V (Mgat5) and the favored ligand for galectins. Herein we report that fibronectin fibrillogenesis and fibronectin-dependent cell spreading are deficient in Mgat5(-/-) mammary epithelial tumor cells and inhibited in Mgat5(+/+) cells by blocking Golgi N-glycan processing with swainsonine or by competitive inhibition of galectin binding. At an optimum dosage, exogenous galectin-3 added to Mgat5(+/+) cells activates focal adhesion kinase (FAK) and phosphatidylinositol 3-kinase (PI3K), recruits conformationally active alpha5beta1-integrin to fibrillar adhesions, and increases F-actin turnover. RGD peptide inhibits PI3K-dependent fibronectin matrix remodeling and fibronectin-dependent cell motility, while galectin-3 stimulates and overrides the inhibitory effects of RGD. Antibodies to the galectin-3 N-terminal oligomerization domain stimulate alpha5beta1 activation and recruitment to fibrillar adhesions in Mgat5(+/+) cells, an effect that is blocked by disrupting galectin-glycan binding. Our results demonstrate that fibronectin polymerization and tumor cell motility are regulated by galectin-3 binding to branched N-glycan ligands that stimulate focal adhesion remodeling, FAK and PI3K activation, local F-actin instability, and alpha5beta1 translocation to fibrillar adhesions.     

Reconstitution of galectin-3 alters glutathione content and potentiates TRAIL-induced cytotoxicity by dephosphorylation of Akt

We investigated the role of galectin-3 in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptotic death in human breast carcinoma BT549 cells. We observed that parental galectin-3 null BT549 cells (BT549(par)) as well as control vector transfected (BT549(neo)) cells were resistant to TRAIL, while galectin-3 cDNA-transfected BT549 cells (BT549(gal-3)) were sensitive to TRAIL. Data from flow cytometry and immunoblotting analyses reveal that reconstitution of galectin-3 promoted cell death and PARP cleavage as well as caspase (-8, -9, and -3) activation during TRAIL treatment. However, unlike TRAIL treatment, galectin-3 transfectants were resistant to UV-B-induced PARP cleavage. Data from cDNA array analysis show that galectin-3 did not significantly enhance or reduce any apoptosis-related gene expression. Moreover, although galectin-3 restored pre-mRNA splicing activity and resulted in elevation of FLIPs protein, experiments with FLIPs cDNA-transfected cells show that overexpression of FLIPs did not sensitize cells to TRAIL. Interestingly, BT549(gal-3) cells demonstrated a approximately 2-fold increase in total glutathione content as well as a approximately 5-fold increase in GSSG content in comparison to BT549(par) and BT549(neo) cells, suggesting that galectin-3 overexpression may alter intraceullular oxidation/reduction reactions affecting the metabolism of glutathione and other thiols. In addition, galectin-3 overexpression inactivated Akt by dephosphorylation. Finally, overexpression of constitutively activated Akt protected BT549(gal-3) cells from TRAIL-induced cytotoxicity. Taken together, our data suggest that galectin-3-enhanced TRAIL-induced cytotoxicity is mediated through dephosphorylation of Akt, possibly through a redox-dependent process.     

结直肠癌中Galectin-3和β-catenin的表达与相关性研究

目的探讨结直肠癌中Galectin-3和β-catenin的表达与临床病理参数之间的关系。方法采用免疫组化En Vision法检测83例结直肠癌组织中galectin-3和β-catenin的表达。结果Galectin-3在结直肠癌中的阳性表达率为81.9%,β-catenin的异常表达率为62.7%。结直肠癌中galectin-3的表达与肿瘤的分化程度、浸润深度、淋巴结转移和病理分期有关(P0.05),而与患者年龄、肿瘤部位、肿瘤大小和脉管侵犯无关(P0.05)。结直肠癌中β-catenin的异常表达与分化程度、淋巴结转移、脉管侵犯和病理分期有关(P0.05),而与患者年龄、肿瘤部位、肿瘤大小和浸润深度无关(P0.05)。结直肠癌中galectin-3的表达与β-catenin异常表达呈正相关(P0.05)。结论Galectin-3的表达可能与结直肠癌的高浸润转移能力有关,其可能是通过β-catenin表达异常而促进肿瘤的浸润扩散。     

Expression of galectin-3 in the rat pancreas during regeneration following hormone-induced pancreatitis

Supramaximal dosage of the cholecystokinin analog caerulein leads to edematous pancreatitis with subsequent acinar cell destruction predominantly by apoptosis. We have used immunohistochemistry to reveal the expression of the anti-apoptotic protein galectin-3 in pancreatic acinar cells. Galectin-3, which occurs only in duct cells under physiological conditions, is expressed in a subset of acinar cells after the end of a 12-h caerulein infusion, giving rise to a patchy staining pattern. During the subsequent period of inflammation and regeneration, galectin-3 expression increases in those acinar cells that undergo apoptosis. By 48 h after the end of caerulein infusion, morphologically normal cells do not contain galectin-3 and participate in regeneration by proliferation. Tubular complexes, being transient structures from degenerative acini, accumulate galectin-3 in the remnants of the epithelium cells. Stimulation with supramaximal dosages of caerulein of the cell line AR4–2J, which is derived from rat pancreatic acinar cells, also results in a marked increase of galectin-3, confirming the in vivo results. We postulate that the high expression of the anti-apoptotic protein galectin-3 regulates the time course of the apoptotic process in pancreatic acinar cells.     

Galectin-3 binds to MUC1-N-terminal domain and triggers recruitment of β-catenin in MUC1-expressing mouse 3T3 cells

Background

Galectin-3 is expressed in a variety of tumors and its expression level is related with tumor progression. Aberrant expression of MUC1 in various tumors is also associated with a poor prognosis. It has been reported that MUC1 is a natural ligand of galectin-3.

Methods

A stable MUC1 transfectant was produced by introducing MUC1 cDNA into mouse 3T3 fibroblasts (MUC1/3T3 cells). MUC1 was prepared from MUC1/3T3 cells; MUC1-N-terminal domain (MUC1-ND) and -C-terminal domain (MUC1-CD) were separated by CsCl ultracentrifugation, and then the galectin-3-binding domain was determined by co-immuniprecipitation assay. After ligation of galectin-3 to 3T3/MUC1 cells, MUC1-CD was immunoprecipitated from the cell lysate. The immunoprecipitate was subjected to SDS-PAGE and Western blotting, followed by detection of co-immunoprecipitated β-catenin.

Results

Galectin-3 binds to the N-terminal domain of MUC1 but not to the C-terminal one. Galectin-3 present on the cell surface increased with the expression of MUC1 and is colocalized with MUC1. It should be noted that β-catenin was detected in the immunoprecipitate with anti-MUC1-CD Ab from a lysate of galectin-3-treated 3T3/MUC1 cells.

Conclusions

Galectin-3 binds to MUC1-ND and triggers MUC1-mediated signaling in 3T3/MUC1 cells, leading to recruitment of β-catenin to MUC1-CD.

General significance

This signaling may be another MUC1-mediated pathway and function in parallel with a growth factor-dependent MUC1-mediated pathway.     

Negative regulation of RPE cell attachment by carbohydrate-dependent cell surface binding of galectin-3 and inhibition of the ERK-MAPK pathway

Adhesion and spreading of retinal pigment epithelial (RPE) cells on fibronectin-rich extracellular matrices is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). In the present study we explored the capacity of galectin-3, a β-galactoside-binding endogenous lectin, to inhibit early PVR-associated cellular events from a therapeutic perspective. We assessed the relative expression levels of galectin-3 in native RPE and dedifferentiated, cultured RPE. Galectin-3 was constitutively expressed under in vivo and in vitro conditions and was abundant in cultured cells. Treatment of human RPE cells with soluble galectin-3 disclosed no toxicity within control limits up to 250 μg/ml. When added to the medium, galectin-3 dose-dependently inhibited attachment and spreading of the cells on fibronectin by more than 75%. When coated on the plastic surface, galectin-3 alone impaired attachment and spreading of RPE cells, and reduced attachment but not spreading on fibronectin. Galectin-3 bound to the cell surface, and, as determined by the use of the competing sugar β-lactose, galectin-3-mediated effects were dependent on carbohydrate binding. To ascertain the role of the ability of galectin-3 to form pentamers, we proteolytically removed the N-terminal, cross-linking section. The remaining C-terminal carbohydrate-binding domain alone failed to bind to cells and was functionally inactive. These results emphasize the relevance of both properties, i.e., glycan-binding and cross-linking of glycan moieties, for the inhibitory activity of galectin-3. Incubation of mobilized RPE cells with galectin-3 significantly disturbed microfilament assembly and, in correlation with decreased attachment, inhibited ERK phosphorylation. Therefore, galectin-3, acting as a cross-linking lectin on the cell surface, negatively regulates attachment and spreading of RPE cells in vitro. This effect, at least in part, is attributed to an inhibition of the ERK-MAPK pathway, which prevents cytoskeletal rearrangements needed for RPE cell attachment and spreading. Further investigation at this pathway may disclose a promising nouveau perspective for treatment and prophylaxis of early PVR.     

Galectin-3 but not galectin-1 induces mast cell death by oxidative stress and mitochondrial permeability transition

Galectin-1 and galectin-3 are the most ubiquitously expressed members of the galectin family and more importantly, these two molecules are shown to have opposite effects on pro-inflammatory responses and/or apoptosis depending on the cell type. Herein, we demonstrate for the first time that galectin-3 induces mast cell apoptosis. Mast cells expressed substantial levels of galectin-3 and galectin-1 and to a lesser extent the receptor for advanced glycation end products (RAGE) on their surfaces. Treatment of cells with galectin-3 at concentrations of > or =100 nM for 18-44 h resulted in cell death by apoptosis. Galectin-3-induced apoptosis was completely prevented by lactose, neutralizing antibody to RAGE, and the caspase-3 inhibitor z-DEVD-fmk. Galectin-3-induced apoptosis was also completely abolished by dithiothreitol and superoxide dismutase, but not inhibited by catalase. Moreover, galectin-3 but not galectin-1 induced the release of superoxide, which was blocked by lactose, anti-RAGE, and dithiothreitol. Finally, galectin-3-induced apoptosis was blocked by bongkrekic acid, an antagonist of the mitochondrial permeability transition pore (PTP), while atractyloside, an agonist of the PTP, greatly facilitated galectin-1-induced apoptosis. These data suggest that galectin-3 induces oxidative stress, PTP opening, and the caspase-dependent death pathway by binding to putative surface receptors including RAGE via the carbohydrate recognition domain.     

Interaction of a novel cysteine and histidine-rich cytoplasmic protein with galectin-3 in a carbohydrate-independent manner

We have used the yeast two-hybrid system to search for cytoplasmic proteins that might assist in the intracellular trafficking of the soluble beta-galactoside-binding protein, galectin-3. We utilised as bait murine full-length galectin-3 to screen a murine 3T3 cDNA library. Several interacting clones were found to encode a partial open reading frame and a full-length clone was obtained by rapid amplification of cDNA ends methodology. In various assays in vitro the novel protein was shown to bind galectin-3 in a carbohydrate-independent manner. The novel protein contains an unusually high content of cysteine and histidine residues and shows significant sequence homologies with several metal ion-binding motifs present in known proteins. Confocal immunofluorescence microscopy of permeabilised 3T3 cells shows a prominent perinuclear, as well as cytoplasmic, localisation of the novel protein.     

Tyrosine-phosphorylated galectin-3 protein is resistant to prostate-specific antigen (PSA) cleavage

Galectin-3 is a chimeric carbohydrate-binding protein, which interacts with cell surface carbohydrate-containing molecules and extracellular matrix glycoproteins and has been implicated in various biological processes such as cell growth, angiogenesis, motility, and metastasis. It is expressed in a wide range of tumor cells and is associated with tumor progression. The functions of galectin-3 are dependent on its localization and post-translational modifications such as cleavage and phosphorylation. Recently, we showed that galectin-3 Tyr-107 is phosphorylated by c-Abl; concomitantly, it was also shown that galectin-3 can be cleaved at this site by prostate-specific antigen (PSA), a chymotrypsin-like serine protease, after Tyr-107, resulting in loss of galectin-3 multivalency while preserving its carbohydrate binding activity. Galectin-3 is largely a monomer in solution but may form a homodimer by self-association through its carbohydrate recognition domain, whereas, in the presence of a ligand, galectin-3 polymerizes up to pentamers utilizing its N-terminal domain. Oligomerization is a unique feature of secreted galectin-3, which allows its function by forming ordered galectin-glycan structures, i.e. lattices, on the cell surface or through direct engagement of specific cell surface glycoconjugates by traditional ligand-receptor binding. We questioned whether Tyr-107 phosphorylation by c-Abl affects galectin-3 cleavage by PSA. The data suggest a role for galectin-3 in prostate cells associated with increased activity of c-Abl kinase and loss of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) activity. In addition, the ratio of phosphorylated/dephosphorylated galectin-3 might be used as a complementary value to that of PSA for prognosis of prostate cancer and a novel therapeutic target for the treatment of prostate cancer.