Study On Correlation Between Sulfation Or Fucosylation Of Glycans And Metastasis

1. Discrepancy expression of lactosylsulfatide between high and low metastasispotential hepatocellular carcinoma cells In this section, comparison study of sulfatide expression was performed between thehepatocellular carcinoma cells with high and low metastasis potentials to observe thepossible relationship between lactosylsulfatide expressional levels and hepatocellularcarcinoma metastasis potential. By means of immunofluorescent staining and cellELISA with specific monoclonal antibodies against sulfatide, lactosylsulfatide, orgalactocerebroside, it was observed that the expression level of sulfatide in highmetastasis hepatocarcinoma cells was significantly higher than that in low metastasishepatocarcinoma cells. This indicated that there was a difference in expression ofsulfatide in hepatocellular carcinoma cells with different metastasis potentials. After the 7复旦大学 博士论文hepatocellular carcinoma cells with high metastatic potential were treated with 10μmol/l retinoic acid, the level of sulfatide on the cell surface was significantly decreased(P<0.05) and close to the level in low metastatic hepatocellular carcinoma cells. Thelevel of galactocerebrosides which was the precursor of sulfate did not changesignificantly after the treatment by retinoic acid. This indicated that the sulfation ofcerebroside was inhibited by retinoic acid treatment. Further by analysis with thespecific monoclonal antibodies against lactosylsulfatide (SM3) and sulfatide(SM3,SM4), the main molecule sulfatide in hepatocellular carcinoma cells waslactosylsulfatide.2. Establishment of high expressional system of sulfatide in hepatocellular carcinomaand melanoma cells In order to observe sulfatide effect on the malignant behaviors of cancer cellssuch as hepatocellular carcinoma cells and melanoma cells, a high expressionalsystem of sulfatide was established in this section. Sulfotransferase usually possessesthe characteristics of highly specific choosing substrate sugar chains. Even highlyexpressed in some cells, the sulfotransferase may not work well because ofinsufficient substrates and the enzyme activity. In this section, an open readingframe of CST (EC2.8.2.11) gene was inserted into the expressional vector pCXN2 atthe cloning sites to construct a CST expressional plasmid. By means ofLipofectAmine transfection, the expressional recombinant vectors were transferred tohuman Hep3B hepatocellular carcinoma cells and B16/F1 melanoma cells. Afterscreening with G418, the transfected cells with Neo marker grew into clonal cells. ByNorthern blot analysis of the total RNA from the transfected cells, a higherexpressional level of CST mRNA was observed in the transfectants, while no manifestexpression was noted in Mock cells which were transfected with pCXN2 vector.This indicated that a higher CST expression cell system was achieved. However,higher expression of CST gene did not mean that cerebroside sulfotransferase workedwell in such cells. Therefore we further analyzed the clonal cells by means of specificmonoclonal antibody detection. Two clones of each transfectants were screened withhigh level of sulfatide which was catalyzed by CST. Hepatocellular carcinoma cellsgot two clones designed as CST 6 和 8,melanoma cells also got two clones as CST 1and 3. To confirm the product further in the transfectants, total acid lipid from thetransfectants was extracted and developed by thin layer chromatograminmmunoblotting and flow cytometry analyses. In results, a higher level of sulfatide 8复旦大学 博士论文was confirmed in the transfectants, while not in the Mock cells. In comparison withthe standard lactosylsulfatide and galactosylsulfatide by monoclonal staining,lactosylsulfatide in the transfectants was the main molecule of sulfatide. The results inthis section suggested that a high expression system of sulfatide was established.3. Lactosyl sulfa...