Function Of Protein O-GlcNAcylation In The Antitumour Activity Of Taxol

O-GlcNAcylation is the addition of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1984 by the famous biochemist Gerald W. Hart. Recent studies have shown that protein O-GlcNAcylation is involved in multiple cellular processes, such as transcription, translation, cell signal transduction and cell stress-response. Abnormal O-GlcNAcylation will lead to many diseases, such as cancer, diabetes and a variety of neurodegenerative diseases. Our early studies found that the expression of protein O-GlcNAcylation significant change after Taxol treatment of breast cancer cells. Therefore, this thesis is aim to study the effects of Taxol on protein O-GlcNAcylation in the antitumor activity.In the thesis, the effects of Taxol on protein O-GlcNAcylation were determined in vivo and in vitro, and the related enzymes in the biosynthesis of O-GlcNAcylation were analyzed to study the mechanism. We also examined whether protein O-GlcNAcylation would affect the anti-proliferation activity of Taxol in breast cancer cells. For further study, HK2 and PKM2, the two key enzymes in the process of glycolysis, were detected with MS to analyze their O-GlcNAc sites. Breast cancer cell lines overexpressing HK2 or PKM2 and cell lines with endogenous HK2 or PKM2 gene silenced were established, respectively.In vitro, the expressions of protein O-GlcNAcylation and related enzymes in the breast cancer cells MDA-MB-231 treated with different concentration of Taxol were determined by using Western blotting. The results showed that Taxol up-regulated the protein O-GlcNAcylation, OGA and OGT expressions with a dose-dependent manner, but there were no significant change on PFK1,PKM2, GFAT1, GLUT1.The results of RT-qPCR showed that mRNA levels of OGA and OGT were dose-depedently increased with Taxol. Western blotting analysis also showed that Taxol time-dependently increased the expressions of protein O-GlcNAcylation, OGA and OGT, but the expressions of PFK1, PKM2, GFAT1, GLUT1 presented no significant change. The data of SRB assay showed that OGA inhibitor TMG or PUGNAc had no significant inhibitory effect on the growth of breast cancer cells MDA-MB-231. Compared with Taxol treatment alone, the combined treatment of TMG or PUGNAc with Taxol did not cause any significant effects on cell growth. As showen in the Western blotting analysis, TMG or PUGNAc up-regulated the expression of protein O-GlcNAcylation in MDA-MB-231. Therefore, the results indicated that O-GlcNAcylation induced by TMG or PUGNAc had no significant influence on the cell growth inhibitory effects of Taxol. In the antitumor experiment of OGT inhibitor alloxan, SRB data indicated that 0-5mM alloxan had no significant growth inhibitory effect on breast cancer cells MDA-MB231, while Western blotting analysis showed alloxan down-regulated protein O-GlcNAcylation. The results of antitumor experiment of OGT inhibitor alloxan indicated that O-GlcNAcylation induced by alloxan had no significant effect on the cell growth inhibitory of Taxol. When breast cancer cells MDA-MB-231 were treated with Taxol, expressions of OGA and OGT in the cytoplasm were increased significantly. Furthermore, we determined the protein O-GlcNAcylation in HCT116 cells expressing different p53 protein levels, and the results demonstrated that p53 gene had relationship with O-GlcNAcylation.In nude mice model, the expressions of protein O-GlcNAcylation, OGA and OGT in the tumor tissue and liver tissue were determined by Western blotting. The analysis data showed that Taxol had no significant effect on the expressions of protein O-GlcNAcylation, OGA and OGT. The results in vivo were different from that in vitro.In the thesis, HK2 and PKM2 were extracted from the cells transfected with certain plasmid, and then enriched by Immunipreicipitation, respectively. The O-GlcNAc sites on HK2 and PKM2 were identified with MS after proteins were pretreated by in-gel digestion. Finally,3 O-GlcNAc sites were detected on HK2 while 16 O-GlcNAc sites were identified on PKM2. These data will process the study of the function of O-GlcNAcylation.For further study, a series of breast cancer cell lines with different characteristics were established. Eukaryotic expression vectors including HK2 or PKM2 gene were transfected by lipofectamine 2000 into breast cancer cells MDA-MB-231, then stable cell lines overexpressing HK2 or PKM2 were established by limited dilution assay. The MDA-MB-231 cell lines with endogenous HK2 or PKM2 gene silenced were constructed with shRNA interference technique.In above, the effects of Taxol on protein O-GlcNAcylation were studied preliminarily in vitro and in vivo. We found that Taxol up-regulated the expression of protein O-GlcNAcylation and the change of O-GlcNAcylation had no significant effect on the anti-proliferation activity of Taxol in breast cancer cells. O-GlcNAc sites on PKM2 and HK2 were identified and a series of breast cancer cell lines as descripted above were constructed. Theses current results will provide foundation for further studies to explore the effects of O-GlcNAcylation in the antitumor activity.