Overexpression of lysosomal-type sialidase leads to suppression of metastasis associated with reversion of malignant phenotype in murine B16 melanoma cells

Abstract

Increased sialylation in cell surface glycoproteins is one characteristic feature of cancer cells, particularly related to their metastatic potential and invasiveness. Expression of lysosomal-type sialidase, which plays a major role in hydrolysis of such sialo-glycoproteins, is therefore considered to have a great influence on malignant properties of cancer cells. To investigate whether the sialidase expression level is linked to the malignant phenotype, we transfected B16-BL6 murine melanoma cells, a highly invasive and metastatic line, with an expression vector harboring a rat lysosomal sialidase cDNA; then clones were isolated and examined for changes in biological character. Sialidase-overexpressing cells showed suppression of experimental pulmonary metastasis and tumor progression. The transfectants exhibited diminished cell growth, anchorage-independent growth and increased sensitivity to apoptosis induced by suspension culture or serum depletion in vitro, but no significant alterations in invasiveness, cell motility and cell attachment to fibronectin, collagen IV and laminin. Flow cytometric analysis with either peanut agglutinin (PNA) or Ricinus communis agglutinin (RCA) lectin revealed that desialylated forms of glycoproteins on the cell surfaces were increased. In particular, a desialylated form of a cell surface glycoprotein of 83 kDa was prominent in the transfectants, as determined by galactose oxidase labeling. These observations indicate that sialidase expression is inversely associated with metastatic potential and tumor growth in cancer cells, probably through a regulation mechanism that suppresses cell growth and anchorage-independent growth and promotes apoptosis with deprivation of cell anchorage. © 2001 Wiley-Liss, Inc. Key words: sialidase; metastasis; anchorage-independent growth; sialic acidsSialidase (EC 3.2.1.18) is a key enzyme in catabolism of glycoproteins and glycolipids. Desialylation of these glycoconjugates is a crucial event leading to modulation of cellular functions in numerous physiological and pathological processes. 1,2 Alterations in sialylation during malignant transformation have been observed to be closely associated with the malignant phenotype in terms of metastatic potential and invasiveness. [3][4][5][6][7] To understand how sialidases are involved in this aberrant sialylation, our study has focused on mammalian forms in cancer cells.Mammalian sialidases are classified into 3 to 4 forms based on their subcellular localization and substrate preference. Cytosolic, lysosomal and membrane forms of the sialidases have been cloned; their primary sequences revealed that they are proteins encoded by distinct genes with different major subcellular locations and enzymatic properties. 8 We previously demonstrated that metastatic potential is inversely correlated with lysosomal-type sialidase activity in transformed rat 3Y1 cells 9 and in murine B16 melanoma cells, 10 whereas it did not have a significant relation to sialic acid levels. We also investigated the role of sialidas...

Sialidase NEU4 Hydrolyzes Polysialic Acids of Neural Cell Adhesion Molecules and Negatively Regulates Neurite Formation by Hippocampal Neurons

“…NEU1 seemed to slightly increase the product up to 3-5% of that of NEU4b with DP2 and DP6, when the sialidase gene was co-transfected with the protective protein (carboxypeptidase A) pp11 gene, known to be an essential factor for sialidase activity together with ␤-galactosidase in the case of the human NEU1 enzyme. It should be noted here that unlike the case with the human NEU1 enzyme, pp11 co-transfection resulted in only a slight increase in sialidase activity even toward 4MU-NeuAc (120% of the activity without pp11), consistent with our previous report of less dependence of protective protein for the rat NEU1 enzyme (27). These results suggest that NEU4 reacts on sialylpolymers and possesses exo-sialidase activity, although the possibility of endo-sialidase activity still remained.…”

“…Sialidase Activity-For sialidase enzymatic studies, HEK293T cells were transiently transfected with the respective expression plasmids for murine sialidase genes Neu2 (21), Neu3 (21), and two splicing isoforms Neu4a and Neu4b (25) and for rat Neu1 gene (27), previously prepared. For NEU1 activity, the protective protein gene (pp11) was sometimes co-transfected (28).…”

NEU1 Sialidase Expressed in Human Airway Epithelia Regulates Epidermal Growth Factor Receptor (EGFR) and MUC1 Protein Signaling

“…4) implicates NEU1 in airway epithelial repair and wound healing, tumorigenesis, and metastatic potential (1,74). In fact, NEU1 overexpression has been reported to suppress anchorage-independent growth and metastasis of murine B16 melanoma cells (75) and human colon cancer cells (76). Because NEU1 desialylates MUC1 (Fig.…”

Developmental Change of Sialidase Neu4 Expression in Murine Brain and Its Involvement in the Regulation of Neuronal Cell Differentiation

“…Lectin Blotting-Lectin blot analyses of SSA (Sambucus sieboldiana agglutinin), MAM (Maackia amurens mutagen), PNA (peanut agglutinin), and RCA (Ricinus communis agglutinin, Honen, Tokyo, Japan) were conducted as described previously (18). Briefly, cell homogenates were resolved on an 8% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes.…”