博士 / 國立陽明大學 / 生物化學研究所 / 93 / Fucosyltransferases (Fuc-Ts) catalyze the transfer of fucose from guanosine diphosphate β-L-fucose (GDP-fucose) to glycans of glycoconjugates (glycoproteins and glycolipids). These fucosylated glycoconjugates not only have a role in providing structural components, but also in mediating cell-cell interactions and cell-microbes interactions. Additionally, many studies have demonstrated that tumor metastasis potential correlated with the elevations of gene expression or activity of Fuc-Ts. Therefore it is necessary to discover the effective fucosyltransferase inhibitors to elucidate the functional role of Fuc-Ts in the complex biological systems. Moreover the inhibitors are potential useful as anti-tumor agents. To date, many strategies of development of Fuc-Ts’ inhibitors was used. The commonest approach is the design of acceptor substrate, donor (GDP-fucose), and transition-state analogues by chemical synthesis. But most of these inhibitors are not cell-permeable, and rarely display effective inhibitory ability in cell models. In the present study, we chose the screening strategy from microbial metabolites to develop new type Fuc-Ts’ inhibitors and observed the influences of these inhibitors on cells.
After screening for 3250 samples of microbial extracts against Fuc-TV, stachybotrydial, an known compound purified from culture broth of the fungus Stachybotrys cylindrospora was discovered to be a potent inhibitor. Meanwhile, four similar compounds, stachybotrylactone、2��-hydroxystachybotrylactone and two new compounds, were purified from the same culture broth. Studying about the inhibition activity of these five compounds for other glycosyl- transferases, we found that stachybotrydial is the strongest inhibitor, and they are not specific inhibitors for Fuc-Ts. They can also inhibit sialyltransferases activity. But they can’t inhibit galactosyltransferases activity. Inhibition kinetic analysis indicated that stachybotrydial is an uncompetitive inhibitor with respect to the donor GDP-fucose and a noncompetitive one with respect to the acceptor substrate LacNAc with Ki of 10.7 and 9.7 μM, respectively.
U937 human leukemia cells treated with stachybotrydial were stained with different lectins or antibodies to detect the change of glycans on cell surface. The fluorescence intensity of the stained cells was analyzed with FACScan flow cytometer. After treatment with 3 μM stachybotrydial for 1 day, the expression of fucose on cell surface of U937 cells significantly decreased to 60% of control cells. Sialic acid content on cell surface was no significantly different from control cells. But galactose content on cell surface increased to 125% of control cells. The tumor markers, sLex and sLea content on cell surface slightly decreased. The result proved that the expression of fucose on U937 cell surface can be inhibited by the Fuc-Ts’ inhibitor, stachybotrydial. We further investigated the adhesion ability of stachybotrydial treated U937 cells to E-selectin. After treatment with 3μM stachybotrydial for 1 day, the number of E-selectin adhesive cells decreased to 60% of control cells. Therefore, stachybotrydial can also inhibit the cell adhesion to E-selectin.
U937 cell would die by treatment with high dose of stachybotrydial. From observation of the alteration of cell and nuclear morphology, the result of cell cycle assay and DNA fragmentation assay, we demonstrated that stachybotrydial can induce cell apoptosis. But we don’t know yet what mechanism would lead cell apoptosis by stachybotrydial.
In conclusion, stachybotrydial is a new type inhibitor of Fuc-Ts. These observations in this study provide the new chemical structure information for the design of stronger inhibitors. This compound also has other physiological activities and is worth to further study.
| Identifer | oai:union.ndltd.org:TW/093YM005107023 |
| Date | January 2005 |
| Creators | Tzu-Wen Lin, 林子文 |
| Contributors | Ying-Chieh Tsai, 蔡英傑 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 0 |
Page generated in 0.0096 seconds