Altered glycosylation patterns have been reported in breast cancer cells but despite some understanding of these changes, the role of glycosidases remains an underresearched area of glycobiology. The aim of this research project was to establish an in vitro model for the biochemical study of glycosidases and their isoforms in normal and cancer breast cells grown in vitro. A glycosidase assay based on paranitrophenol sugar substrates was adapted, and for this purpose showed that there was a significant increase (p<0.05) in a-fucosidase, f3-mannosidase, f3-N-acetylgalactosaminidase and f3-N-acetylglucosaminidase in the breast cancer cell lines compared to the normal breast cell line but the optimum pH for maximum activity was similar in all the five cell lines. The kinetic analysis showed a statistically significant (p<0.05) two-fold increase in Vmax for f3-N-acetylglucosaminidase in the cancerous cells compared to the normal breast cell line but the Km remained unaltered. Two isoforms of f3-N-acetylgalactosaminidase and f3-N-acetylglucosaminidase were isolated from the cell lysate using ion exchange chromatography and no significant differences were found between their pH optima and the enzyme kinetics. The correlation between glycosidase activity and lysosome number in the normal and cancerous cell lines was studied using DND 99 Iysotracker dye and confocal microscopy. The results showed a two fold increase in lysosome number in the MDA MB 435 and MCF 7 compared with the HB4a cells. The activity of the glycosidases secreted into the media surrounding the cells, after 48h incubation, also showed a two-fold increase (p< 0.05) in f3-N-acetylgalactosaminidase and f3-N-acetylglucosaminidase activity in the MDA MB 435 and MCF 7 compared with the HB4a cells. Further treatment of MDA MB 435 cells seeded on Matrigel™ material, with imino sugar and protease inhibitors resulted in a significant 86 percent decrease in secreted f3-N-acetylglucosaminidase activity and a 70 percent decrease in cell adhesion and a decrease in cell invasion in this model system. This is the first time that intracellular and secreted glycosidase activity has been studied in a systematic manner in the above breast cell lines. The Iysotracker data suggests that there is a correlation between the number of Iysosomes and cell lysate glycosidase level. The decrease in MDA MB 435 cell migration through the extracellular Matrigel™ material suggests that glycosidases have a functional role in cancer cell migration.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:502401 |
| Date | January 2008 |
| Creators | Ramessur, Kushen Teewary |
| Publisher | University of Westminster |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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