Galectin-3 regulates p21 stability in human prostate cancer cells.

Galectin-3 (Gal-3) is a multifunctional protein involved in cancer through regulation of cell adhesion, cell growth, apoptosis and metastasis, while p21 (Cip1/WAF1) is a negative regulator of the cell cycle, involved in apoptosis, transcription, DNA repair and metastasis. The results presented here demonstrate for the first time that the level of Gal-3 protein is associated with the level of p21 protein expression in human prostate cancer cells and the effects of Gal-3 on cell growth and apoptosis were reversed by modulating p21 expression level. Furthermore, Gal-3 regulates p21 expression at the post-translational level by stabilizing p21 protein via the carbohydrate-recognition domain. This is the first report suggesting a molecular function not yet described for Gal-3 as the regulator of p21 protein stability. This study provides a unique insight into the relationship of these two molecules during prostate cancer progression, and may provide a novel therapeutic target.

Calpain activation through galectin-3 inhibition sensitizes prostate cancer cells to cisplatin treatment.

Prostate cancer will develop chemoresistance following a period of chemotherapy. This is due, in part, to the acquisition of antiapoptotic properties by the cancer cells and, therefore, development of novel strategies for treatment is of critical need. Here, we attempt to clarify the role of the antiapoptotic molecule galectin-3 in prostate cancer cells using siRNA and antagonist approaches. The data showed that Gal-3 inhibition by siRNA or its antagonist GCS-100/modified citrus pectin (MCP) increased cisplatin-induced apoptosis of PC3 cells. Recent studies have indicated that cisplatin-induced apoptosis may be mediated by calpain, a calcium-dependent protease, as its activation leads to cleavage of androgen receptor into an androgen-independent isoform in prostate cancer cells. Thus, we examined whether calpain activation is associated with the Gal-3 function of regulating apoptosis. Here, we report that Gal-3 inhibition by siRNA or GCS-100/MCP enhances calpain activation, whereas Gal-3 overexpression inhibits it. Inhibition of calpain using its inhibitor and/or siRNA attenuated the proapoptotic effect of Gal-3 inhibition, suggesting that calpain activation may be a novel mechanism for the proapoptotic effect of Gal-3 inhibition. Thus, a paradigm shift for treating prostate cancer is suggested whereby a combination of a non-toxic anti-Gal-3 drug together with a toxic chemotherapeutic agent could serve as a novel therapeutic modality for chemoresistant prostate cancers.

Down-regulation of galectin-3 suppresses tumorigenicity of human breast carcinoma cells.

Galectin-3 is an endogenous beta-galactoside-binding protein with specificity for type I and II ABH blood group epitopes and poly-N-acetyllactosamine glycan-containing cell surface glycoproteins and is the major nonintegrin cellular laminin-binding protein. Galectin-3 is expressed at an elevated level in a wide range of neoplasms, and expression was shown to be associated in some tumor cell systems with metastases. Here we determined the functional consequence of blocking galectin-3 expression in highly malignant human breast carcinoma MDA-MB-435 cells. Inhibition of galectin-3 expression led to reversion of the transformed phenotype as determined by altered morphology, loss of serum-independent growth, acquisition of growth inhibition properties by cell contact, and abrogation of anchorage-independent growth. The blockage of galectin-3 expression led to a significant suppression of tumor growth in nude mice. These results provide direct evidence that galectin-3 expression is necessary for the maintenance of the transformed and tumorigenic phenotype of MDA-MB-435 breast carcinoma cells.

Galectin-3 binding and metastasis.

Galectin-3 (gal-3) is a member of a growing family of carbohydrate-binding proteins. It consists of two functional domains: an amino-(N)-terminal domain, which is cleavable by collagenases and is responsible for dimerization as well as secretion of the protein, and a carboxy-(C)-terminal domain with affinity for carbohydrates containing N-acetylactosamine residues. Gal-3 is present in the nucleus, the cytoplasm, and also the extracellular matrix of many normal and neoplastic cell types. However, an array of reports show an up-regulation of this protein in transformed and metastatic cell lines (1,2). Moreover, in many human carcinomas, an increased expression of gal-3 correlates with progressive tumor stages (3,6).

Galectin-3 induces endothelial cell morphogenesis and angiogenesis.

Increasing evidence suggests that carbohydrate-binding proteins play an essential role in tumor growth and metastasis. However, conflicting results on their function in the regulation of cell proliferation and differentiation during angiogenesis have been reported. We have examined the role of galectin-3 in the regulation of human umbilical vein endothelial cell proliferation, differentiation, migration, and neovascularization. Galectin-3, a carbohydrate-binding protein, with specificity for type 1 and 11 ABH blood group epitopes and polylactosamine glycan containing cell surface glycoproteins, is the major nonintegrin cellular laminin-binding protein. Because galectin-3 expression was shown to be associated in some tumor systems with metastasis, we questioned whether it induces endothelial cell morphogenesis. Here we show that galectin-3 affects chemotaxis and morphology and stimulates capillary tube formation of HUV-EC-C in vitro and angiogenesis in vivo. Endothelial cell morphogenesis is a carbohydrate-dependent process, as it is neutralized by specific sugars and antibodies. These findings demonstrate that endothelial cell surface carbohydrate recognition event(s) can induce a signaling cascade leading to the differentiation and angiogenesis of endothelial cells.

Carbohydrate-recognition and angiogenesis.

Angiogenesis is required for the continual growth of the tumor and provides a gateway for cells to escape the confines of the primary tumor. Angiogenic stimulus triggers a cascade of functional responses leading to local basement membrane dissolution, endothelial cell migration, proliferation and microvessel morphogenesis. In this commentary, we review the significance of carbohydrate-binding proteins involved in angiogenesis. The importance of carbohydrate-recognition processes to angiogenesis stems from the observation that angiogenic factors like fibroblast growth factor family and vascular endothelial growth factors bind initially to the extracellular matrix proteoglycans before binding to their cognate receptors, and some of the adhesion molecules bind to glycoconjugates present on the surface of the endothelial cells. The possible significance of these interactions will be discussed.

The NH2 terminus of galectin-3 governs cellular compartmentalization and functions in cancer cells.

Galectin-3 is a member of the beta-galactoside-binding protein family shown to be involved in tumor progression and metastasis. It has a unique primary structure consisting of three domains: a 12-amino acid leader sequence containing a casein kinase I serine phosphorylation site, which is preceded by a collagenase-sensitive Pro-Gly-rich motif, and a COOH-terminal half encompassing the carbohydrate-binding site. To study the functional role of the unusual leader sequence of galectin-3, a mutant cDNA that causes an 11-amino acid deletion in the NH2-terminal region was generated and expressed in galectin-3-null BT-549 human breast carcinoma cells. Deletion of the NH2 terminus resulted in abolition of the secretion of truncated galectin-3, loss of nuclear localization, and reduced carbohydrate-mediated functions compared with the wild-type protein. When green fluorescent protein was fused to the galectin-3 leader sequence and transiently transfected into BT-549 cells, the uniform cellular distribution of native green fluorescent protein was changed mainly to a nuclear pattern. To further investigate whether the functional changes observed in a galectin-3 with the 11 NH2-terminal amino acids deleted were due to loss of phosphorylation at Ser6, two point mutations were created at this serine: Ser6-->Ala and Ser6-->Glu. No obvious difference was observed in cellular localization between wild-type and Ser6-mutated transfectants. These results suggest a structural role for the NH2 terminus leader motif of galectin-3 in determining its cellular targeting and biological functions independent of phosphorylation.

Galectin-3 and L1 retrotransposons in human breast carcinomas.

Galectin-3 is a galactoside binding protein found at elevated levels in a wide variety of neoplastic cells and thought to be involved in cognitive cellular interactions during transformation and metastasis. Previously, we have shown that introduction of human galectin-3 (Mr 31,000) cDNA into the human breast cancer cells BT-549 which are galectin-3 null and non-tumorigenic in nude mice resulted in the establishment of four galectin-3 expressing clones. Three of them acquired tumorigenicity when inoculated in the mammary fat pad of nude mice. Here, we questioned what is the molecular difference between the nude mouse tumorigenic and non-tumorigenic galectin-3 expressing BT-549 cell clones. Differential display analysis and Northern blotting revealed that, unlike the tumorigenic clones, neither the parental cells nor the non-tumorigenic clone expressed a 6.5 Kb transcript. A 607 bp PCR (polymerase chain reaction) product from the differentially displayed mRNA revealed a 93% sequence homology with the human L1 retrotransposon previously suggested to play a role in the pathobiology of some breast cancers. In addition, we show that the two gene products, i.e., galectin-3 and L1, are co-expressed in breast carcinoma specimens and in other nude mouse tumorigenic cell lines.

Metastasis of human colon cancer is altered by modifying expression of the beta-galactoside-binding protein galectin 3.

BACKGROUND & AIMS: Galectin 3 is a beta-galactoside-binding protein whose expression has been correlated with advanced tumor stage in the colon, but direct evidence for a role in metastasis is lacking. The current study was designed to more directly establish the role of galectin 3 in colon cancer metastasis. METHODS: Galectin 3 levels were manipulated in human colon cancer cells using eukaryotic expression constructs designed to express the complete galectin 3 complementary DNA in either the sense or antisense orientation. Liver colonization was assessed in athymic mice after splenic-portal inoculation or after spontaneous metastasis during cecal growth. RESULTS: Introduction of galectin 3 antisense into metastatic colon cancer cells (LSLiM6, HM7) resulted in a significant reduction in galectin 3-specific messenger RNA and total and cell surface galectin 3 protein. Conversely, stable integration of galectin 3 in the sense orientation resulted in an increase in cellular and cell surface galectin 3 in cells of low metastatic potential (LS174T). Reduction in galectin 3 levels was associated with a marked decrease in liver colonization and spontaneous metastasis by LSLiM6 and HM7 cells, whereas up-regulation of galectin 3 resulted in increased metastasis by LS174T cells. CONCLUSIONS: This study provides direct evidence that galectin 3 plays an important role in colon cancer metastasis.

Direct action of estrogen on sequence of progression of human preneoplastic breast disease.

We have used the MCF10AT xenograft model of human proliferative breast disease to examine the early effects of estradiol exposure on morphological progression of preneoplastic lesions and to define the step(s) in the morphological sequence at which estrogen may act. The effects of estradiol on neoplastic progression of estrogen-receptor-positive MCF10AT cells in the orthotopic site were examined in ovariectomized female nude mice that received subcutaneous administration of implants of 17beta-estradiol or placebo pellets. At 10 weeks, histological analysis of the lesions derived from the estrogen-supplemented group revealed that 92% of lesions displayed histological features of atypical hyperplasia, carcinoma in situ, or invasive carcinoma, and the remaining 8% exhibited histological features of moderate hyperplasia. These highly proliferative lesions are in marked contrast to the control group in which 60% of samples displayed no evidence of hyperplasia. In contrast with control xenografts, estrogen-exposed xenografts demonstrated extensive areas of papillary growth, adenosis-like areas, prominent host inflammatory infiltration, and angiogenesis. Our results suggest that estrogen exerts a growth-promoting effect on benign or premalignant ductal epithelium by enhancing 1) the frequency of lesion formation, 2) the size of lesions, 3) the speed of transformation from normal/mild hyperplasia to those with atypia, 4) the degree of dysplasia, and 5) angiogenesis.

Galectin-3: a novel antiapoptotic molecule with a functional BH1 (NWGR) domain of Bcl-2 family.

Galectin-3, a beta-galactoside-binding protein, has been shown to be involved in tumor progression and metastasis. Here, we demonstrate that expression of galectin-3 in human breast carcinoma BT549 cells inhibits cis-diamminedichloroplatinum (cisplatin)-induced poly(ADP-ribose) polymerase degradation and apoptosis, without altering Bcl-2, Bcl-X(L), or Bax expressions. Galectin-3 contains the NWGR amino acid sequence highly conserved in the BH1 domain of the bcl-2 gene family, and a substitution of glycine to alanine in this motif abrogated its antiapoptotic activity. Our findings demonstrate that galectin-3 inhibits apoptosis through a cysteine protease pathway and highlight the functional significance of the NWGR motif in apoptosis resistance of a non-Bcl-2 protein.

Galectin-3 is a novel substrate for human matrix metalloproteinases-2 and -9.

The primary structure of galectin-3, a approximately 30 kDa galactoside-binding protein (aka CBP-35, mL-34, hL-31, L-29, Mac-2, and epsilon BP), reveals two structural domains: an amino-terminal domain consists of a Pro-Gly-rich motif, and a globular carboxyl-terminal domain containing a carbohydrate-binding site. In this study, we report that the amino-terminal domain of galectin-3 contains a cleavage site for two members of the matrix metalloproteinase family of enzymes: the 72 kDa (gelatinase A, MMP-2) and the 92 kDa (gelatinase B, MMP-9) proteinases. The major cleavage site for the gelatinases in galectin-3 is at the Ala62-Tyr63 bond, and its hydrolysis by these enzymes was inhibited by TIMP-2. Cell-surface expression of galectin-3 was reduced following treatment of viable T47D human breast carcinoma cells with gelatinase A. These results suggest that galectin-3 may be a substrate for gelatinases and that its degradation may play a role in modulating the biological activities of galectin-3.

Regulation of the expression of galactoside-binding lectin during human monocytic differentiation.

The widely distributed hL-31 (CBP35, epsilon BP, mL-34, L-29, Mac-2) is a Ca(2+)-independent galactoside-binding lectin which functions as a receptor on mammalian cells for glycoproteins containing poly-N-acetyllactosamine side chains. Little is known about the regulation of its expression. The human promyelocytic leukemia cell line, HL-60, was used to determine whether expression of hL-31 (Mac-2) correlated with macrophage/monocyte differentiation. Nondifferentiated HL-60 cells and HL-60 cells grown in the presence of 1.24 microM retinoic acid expressed only trace amounts of hL-31. In contrast, both hL-31 transcripts and protein were detected at 8 h after addition of 17 nM 12-O-tetradecanoylphorbol-13-acetate and reached maximal levels at 24 h. Addition of actinomycin D along with 12-O-tetradecanoylphorbol-13-acetate blocked accumulation of hL-31 mRNA. In contrast, addition of actinomycin D to 12-O-tetradecanoylphorbol-13-acetate-treated HL-60 cells that had already accumulated high levels of hL-31 mRNA did not cause significant reduction in RNA levels until 6-8 h had elapsed. Since increased hL-31 expression was not associated with an increase in transcriptional activity of the hL-31 gene, these results suggest that hL-31 expression is regulated at the posttranscriptional level, at least in part, by stabilization of its mRNA. The results also indicate that the processes leading to increased hL-31 expression in HL-60 cells may be specific to differentiation along the monocyte/macrophage pathway.