| Carbohydrate structures with a 3'-sulfoβGal linkage, such as 3'-sulfo-Lex, have been thought to play an important role in many biological processes including cell adhesion, inflammation, and cancer metastasis. Gal:3-O-sulfotransferase-2 (Gal3ST-2) is the key enzyme in the biosynthesis of 3'-sulfo-Lewis antigens. Gal3ST-2 was a newly cloned sulfotransferase whose gene was reported on human chromosome 2q37.3, which transferred sulfate group from the sulfate donor 5'-phosphoadenosine 3'-phosphsulfate (PAPS) to the 3'hydroxyl group of terminal galactose on type I and type II oligosaccharides of glycoproteins, but its biological significance remained unknown. The purpose of our study is to elucidate the effect of Gal3ST-2 and 3'-sulfo-Lex on adhesion and proliferation of hepatocarcinoma cells.Part1 Role of Gal3ST-2 and 3'-sulfo-Lex in regulating cell adhesionTo investigate the role of Gal3ST-2 and 3'-sulfo-Lex in human SMMC-7721 hepatoma cells, we depleted Gal3ST-2 with siRNA and added extrinsic Lewis-x trisaccharide 3'-sulfatesodium salt in cells. After marked decrease of Gal3ST-2 mRNA identified by RT-PCR, Gal3ST-2 siRNA transfected cells showed a striking morphological change from polygon to shuttle shape and lower adhesion ability to culture bottle wall so as to be trypsinized more easily than control cells. Gal3ST-2 silencing by siRNA markedly inhibited the expression of integrin subunit aV, but integrin subunitβ3 almost had no change by Western blot and immunofluorescence as affection of homological CST (cerebroside sulfotransferase) on integrins. Further biochemical analysis revealed the down-regulation of aV expression accompanied with a significantly lower affinity to vitronectin (VN). Lewis-x trisaccharide 3'-sulfatesodium salt could increase the expression of aV at the mRNA and protein level and showed stronger adhesion to VN by a binding assay. The similar effects were observed when Gal3ST-2-silenced cells were reexposed to Lewis-x trisaccharide 3'-sulfatesodium salt. Part 2 Role of Gal3ST-2 and 3'-suIfo-Lex in regulating cell proliferation and apoptosisTo further investigate mechanism of Gal3ST-2 and 3'-sulfo-Lex regulation, Gal3ST-2 siRNA expression plasmids and Lewis-x trisaccharide 3'-sulfatesodium salt were introduced into SMMC-7721 cells (human hepatocarcinoma cell line). When the plasmid was transfected for 48h, the cell growth was inhibited with a trend toward an increase in apoptosis and decrease in proliferation. Our results showed that transfection with Gal3ST-2 siRNA plasmids could suppress expression levels of ILK, decrease phosphorylation levels of PKB/Akt and ERK. Lewis-x trisaccharide 3'-sulfatesodium salt stimulated the expression of ILK, up-regulated phosphorylation of PKB/Akt and ERK. The similar effects were observed when Gal3ST-2-silenced cells were reexposed to Lewis-x trisaccharide 3'-sulfatesodium salt. We also observed that glycogen synthase kinase-3β(GSK-3β) is constitutively active in transfected cells where it phosphorylatesβ-catenin at Ser33, Ser37, and Thr41, targetingβ-catenin for rapid degradation. However, Gal3ST-2 siRNA transfection increased the expression of E-cadherin and decreased tyrosine phosphorylation ofβ-catenin. Lewis-x trisaccharide 3'-sulfatesodium salt-induced inhibition of GSK-3βwhich allowedβ-catenin to accumulate in the cytoplasm and then translocate to the nucleus, either in control cells or in Gal3ST-2 siRNA transfected cells, where it promoted the transcription of genes such as c-myc and cyclin D1. In cancer development, the resistance of cells to apoptosis is one of the most crucial steps. Thus, we also demonstrated that down-regulation ofαV in Gal3ST-2 siRNA transfected cells was associated with a decrease in expression of an antiapoptotic proteins, Bcl-2, with a consequent decrease in the ratios for Bcl-2:Bax. By reexposure to Lewis-x trisaccharide 3'-sulfatesodium salt, the apoptotic response of cells was inhibited and Bcl-2 expression was up-regulated, as well as ratio of Bcl-2/Bax.
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