| O-linked N-acetylglucosamine(O-GlcNAc)is a post-translational modification that involves the attachment of a single beta-N-acetylglucosamine moiety to serine or threonine residues in nuclear and cytoplasmic proteins.The level of cellular O-GlcNAc is regulated by two enzymes:O-GlcNAc transferase(OGT)and O-GlcNAcase(OGA),which are involved in GlcNAc addition and removal modification,respectively.Changes in the extracellular environment can cause rapid changes in the level of O-GlcNAc,however,O-GlcNAcylation of the protein quickly returns to baseline levels after stimulation is removed.The process of O-GlcNAc homeostasis appears to be crucial for the regulation of many cell functions including cell cycle progression,stress response and gene transcription.Destruction of O-GlcNAc homeostasis is thought to lead to the development of diseases such as cancer,diabetes and Alzheimer's disease.In our study,we choosed cisplatin,doxorubicin,and vincristine to respectively act on lung cancer cell H1299,hepatoma cell HepG2,and breast cancer cell MCF-7.As a result,the protein O-GlcNAcylation in tumor cells significantly increasedwith a certain regularity.Therefore,in this dissertation,we studied the cisplatin-induced changes in the glycosylation of O-GlcNAc proteins and their mechanisms,and investigated the effect of O-GlcNAc glycosylation on the antitumor activity of cisplatin.Since changes in the level of protein O-GlcNAcylation are mainly regulated by OGT/OGA and donor UDP-GlcNAc,the effect of cisplatin on the changes in OGT/OGA activity,protein expression and mRNA levelswas studied in this paper.Meanwhile,we also studied the effect of cisplatin on donor UDP-GlcNAc,ATP and the energy metabolism.The effect of cisplatin on the expression profile of O-GlcNAcylation of protein was studied.At the same time,by using inhibitors,the effect of the change of protein O-GlcNAcylation levels on the antitumor effect of cisplatin was studied in vitro and in vivo.In vitro experiments,the effects of the three anticancer drugs cisplatin,doxorubicin and vincristine on cancer cells H1299,HepG2 and MCF-7 were detected by western blotting.As a result,it was found that the level of protein O-GlcNAcylation was significantly increased in a concentration-dependent manner.We choosed different concentrations of cisplatin 0,2,8 and 16 ?g/mL to act on lung cancer cell H1299,the expression levels of OGA,OGT,PFK1,PKM2,GFAT1 and other related proteases had no significant difference.The expression levels of HK2,GRP78 and CHOP decreased significantly with increasing concentrations of cisplatin in a concentration-dependent manner.By RT-qPCR experiments,it was found that the mRNAlevels of OGA and OGT increased significantlywithconcentration-dependent increase of cisplatin concentration.Through OGA and OGT activity experiments,it was found that as the concentration of cisplatin increased,the activity of OGA decreased significantly and was concentration-dependent,while the activity of OGT did not change significantly.The results showed that the decrease of OGA activity is one of the reasons for the increased protein O-GlcNAcylationinducedcisplatin.The sulforhodamine B(SRB)colorimetric assay was used to determine the effect of cisplatin alone and in combination with OGA inhibitor TMG/PUGNAc on the proliferation of lung cancer cell line H1299.The results showed that cisplatin had a significantinhibitory effect on proliferation of lung cancer cells in a concentration-dependent manner.Compared with cisplatin alone,cisplatin combined with 10 ?M TMG or 100 ?M PUGNAc had no significant change on the inhibition of the proliferation of lung cancer cell H1299.Apoptosis was detected by flow cytometry,cisplatin at the concentrations of 0,8,16 and 64 ?g/mL was used to act on H1299 cells,apoptotic rates were 7.48%,13.88%,26.97%and 53.06%,respectively,when cisplatin was combined with 10 ?M TMG,the apoptotic rates of the cells were 9.55%,14.04%,24.65%and 44.8%,respectively.When cisplatin was combined with 100 ?M PUGNAc,the apoptotic rates were 16.12%,23.68%,30.07%and 50.4%,respectively.The results showed that compared with the cisplatin alone group,the apoptosis rate of cisplatin combined with TMG or PUGNAc had no significant change.The results of TMG or PUGNAc showed that the increase of protein O-GlcNAcylation level caused by TMG or PUGNAc did not affect the inhibition of cell proliferation by cisplatin,and it also had no effect on the cell apoptosis induced by cisplatin.The effect of cisplatin alone and in combination with OGT inhibitor Alloxan on the proliferation inhibition of lung cancer cell H1299 was detected by SRB assay.The results showed that compared with cisplatin alone,cisplatin combined with 10 mM Alloxan had no significant change on the inhibit of the proliferation of lung cancer cell H1299,cisplatin at the concentrations of 0,8,16 and 64 ?g/mL was used to act on H1299 cells,apoptotic rates were 7.48%,13.88%,26.97%and 53.06%,respectively.When cisplatin was combined with 10 mM Alloxan,the apoptotic rates of cells were 19.53%,34.26%,55.02%and 74.67%,respectively.The apoptotic rate of combined administration was significantly higher than that of cisplatin alone.The results showed that the OGT inhibitor Alloxan did not affect the cell proliferation inhibitory activity of cisplatin after reducing the level of protein O-GlcNAcylation.Through the relationship between cisplatin-induced protein O-GlcNAcylation level and intracellular UDP-GlcNAc content determined by HPLC,we found thatthe UDP-GlcNAc content increasedfrom 1.9 mmol/1011cells to 4.34 mmol/1011 cells in a concentration-dependent manner with increasing concentration of cisplatin in lung cancer cell H1299.At the same concentration of cisplatin,the UDP-GlcNAc content increasedfrom 0.65 mmol/1011 cells to 4.34 mmol/1011 cells in a time-dependent manner.When GFAT1 inhibitor DON was applied to lung cancer cells,the HPLC assay showed that the UDP-GlcNAc content in the cells decreased from 1.77 mmol/1011cells to 0.88 mmol/1011cells in a concentration-dependent manner as the DON concentration increased.While the DON concentration was fixed at 40 ?M,the UDP-GlcNAc content in the cells decreased from 1.33 mmol/1011cells at 0 hour to 0.61 mmol/1011cells at 48 h with the time-dependent manner.Compared with the cisplatin alone,the level of protein O-GlcNAcylationwas significantly reduced witha concentration-dependent manner in the cells treated withcisplatin in combination with DON.The level of protein O-GlcNAcylation in cells treated with cisplatin in combination with glucose/glutamine was detected by western blotting.The results showed that the level of protein O-GlcNAcylation in cells treated with cisplatin in combination with glucose/glutamine significantly increased compared with the cisplatin alone.The results showed that the concentration of UDP-GlcNAc affected the increase of cisplatin-induced protein O-GlcNAcylation.When H1299 cells were treated with different concentrations of cisplatin,the intracellular ATP content increased from 0.33 mmol/104cells to 0.73 mmol/104cells,and the intracellular glucose content increased from 2.43 ?mol/109cells to 4.83?mol/109cells.The intracellular pyruvate content increased from 1.6 ?g/107cells to 3.8?g/107cells and all in a concentration-dependent manner.The mechanism between energy metabolism and elevated cisplatin-induced protein O-GlcNAcylationremains to be further studied.We further examined the changes in expression profiles of cisplatin-induced O-GlcNAcylation proteins such as triose phosphate isomerase,pyruvate kinase and actin-binding protein2 and the degree of changes in glycosylation by mass spectrometry.The results showed the glycosylated protein was enriched by immunoprecipitation using O-GlcNAc antibody.The results of mass spectrometry showed that some proteins were only expressed in the control group,some proteins were only expressed in the cisplatin administration group,and some proteins were expressed but expressed in both groups.There are differences in levels.In vivo experiments,we used 4 mg/kg cisplatin,8 mg/kg cisplatin,1 mg/kg PUGNAc,the combination 4 mg/kg cisplatin with 1 mg/kg PUGNAc to perform the experiment.During the period of action,the tumor volume of the nude mice was measured and the tumor inhibition rates of these four groups were 13%,83%,5%and 14%,respectively.The level of protein O-GlcNAcylation and the expression levels of OGA and OGT in tumor tissue,lung tissue,and liver tissue were detected by western blotting.The results found thatthe level of protein O-GlcNAcylation and the level of OGT had no significant change in H1299 cells treated with 4 mg/kg and 8 mg/kg cisplatin in these three tissues.The expression of OGA in the tumor tissues increased significantly with the concentration of cisplatin witha concentration-dependent manner,while the level of protein O-GlcNAcylation significantly increased in the cells treated with PUGNAc alone in liver and tumor tissues.The results showed that cisplatin did not change the protein O-GlcNAcylation level of tumor tissue in nude mice.OGA inhibitor PUGNAc can increase the protein O-GlcNAcylation of tumor tissue.However,PUGNAc alone had no inhibitory effect on tumor growth,and the use of PUGNAc in combination with cisplatin did not alter the inhibitory effect of cisplatin on tumor growth.In summary,the present study concludes that anti-tumor agents increased the level protein O-GlcNAcylation,and the level protein O-GlcNAcylation induced by cisplatin is elevated mainly by increase of OGA activity and increase of UDP-GlcNAc concentration,increased or decreased protein O-GlcNAcylation levels did not alter the cisplatin inhibitory activity on cell proliferation and also can't alterthe inhibitory activity of cisplatin on tumor growth innude mice.This study has laid a good foundation for solving the cisplatin resistance and improving the sensitivity of tumor cells to cisplatin in the direction of protein O-GlcNAcylation... |
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