Screening and Purification of Sialyltransferase Inhibitors and Effects on Tumor Cell

• Main Authors: Chi-Cheng Hsu, 徐啟正
• Other Authors: Ying-Chieh Tsai
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/01671272758193628047

博士 === 國立陽明大學 === 生化暨分子生物研究所 === 94 === Sialic acids belong to a nine-carbon amino sugar neuraminic acid family, and they are widely distributed in nature as the terminal sugars of oligosaccharide chains of glycoconjugates (glycoproteins and glycolipids). Recently, it has been demonstrated that the sialylation on cell surface is involved in biological and pathological processes, such as differentiation, oncogenic transformation and tumor metastases and invasions. Cell sialylation is dependent on the sialyltransferase which catalyst the transfer of sialic acid from cytidine-5-monophospho-N-acetylneuramic acid (CMP-Neu5Ac) to the terminal position of oligosaccharide chain of glycoconjugate. Therefore, modulation of the sialyltransferase (ST) activity has become an interesting and important subject in the elucidation of the roles played by ST either in normal or pathological processes. In 1980s, although the ST inhibitors based on the analogs of donor substrate were successfully synthesized, these compounds showed no substrate specificity and could not be used in clinical application. Recently, lead compounds based on the transition-state structure of CMP-Neu5Ac have been tested successfully and achieved good inhibition in vitro. However, these transition-state structure-based inhibitors are cell-impermeable and not suitable for in vivo applications. Therefore, in this thesis, we tried to find out new ST inhibitors from natural products and microbial metabolites for potential in vivo application. Sixty-two candidates were screened out from 3520 samples and 9 strains of fungi, 10 strains of bacteria, and 9 strains of actinomyces were obtained. We also tentatively tried to purify the ST inhibitor from the fungus strain 132359 and about 3 g of crude product was successfully obtained from the culture media by using the submerged fermentation method. In 2001, we discovered a new ST inhibitor, soyasaponin I (SsaI), from the soybean extract. In this thesis, the effects of SsaI on the metastatic and invasive behaviors of several cancer cells were also studied. At first, the cytotoxic effect of SsaI on cancer cells were investigated and the results showed that SsaI displayed significant growth inhibition activity only at concentration greater than 100 μM. Then, the ST enzyme activities of various cancer cell lines were measured and it showed that MCF-7 cell has high ST3Gal I enzyme activity; therefore, the MCF-7 cell were chosen for further analysis. The α2,3-ST activity in cellular homogenate prepared from MCF-7 cell could be inhibited by SsaI. When added in culture medium, the SsaI not only reduced the amount of α2,3-sialic acid on MCF-7 cell surface in a dose-dependent manner but also stimulate the adhesion of MCF-7 cell to both type I collagen and the Matrigel-matrix. SsaI also significantly decreased the migration ability of the highly metastastic MDA-MB-231 cell. All together, these studies suggested that SsaI could potentially be used to affect the invasive behavior of tumor cells by modulating the activity of ST. These data suggest that de-regulated α2,3-sialylation could played a crucial role in the invasion and metastases of tumor cells. SsaI is a good candidate for studying the biological roles of ST, and might provide a new preventive strategy in tumor metastasis.