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Chemo-enzymatic Synthesis of Cellular Adhesion Motif RGD and Its Effects of Inhibiting Growth of Tumour Cells and Inducing Apoptosis

Author: WangHua
Tutor: ZhangXueZhong
School: Jilin University
Course: Biochemistry and Molecular Biology
Keywords: RGD Peptide synthesis Trypsin Organic solvents Apoptosis Enzymatic synthesis of HCT-8 Derivative
CLC: R730.5
Type: PhD thesis
Year: 2007
Downloads: 230
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Abstract


Many proteins present in extracellar matrix(ECM)and blood plasma,contain a common tripeptide amino acid sequence Arg-Gly-Asp(RGD)as a recognition site for cellular adhesion,spreading and motility of cells. The RGD motif plays a key role in mediating integrin-matrix interaction and signals transmission,which modulate cell-survival and function. RGD and some synthetic RGD-containing peptides as competitive, reversible inhibitors for adhesive proteins binding have been widely used to study adhesive interaction between cells and suppress tumor metastasis etc. which is the further extension of the RGD, have been reported to make these sequences be even more efficient adhesion motifs to integrins of cell membrane. For example,RGDS-NH2 peptide exhibits additional potent anti-chemotactic and pro-apoptotic effects independently from its anti-adhesive action. Recently, it has been also reported that RGD can enter the cells directly and induce apoptosis by activating some members of caspase family, likely by entering the cells and directly activating caspase 8 and 9,and lately caspase 3,implying unexpected intracellular actions of the RGD-motif. It attracts more and more attention of researchers on studying the effect of RGD on inducing apoptosis of tumor cells. Therefore, the RGD-containing peptides are looking forward to treat some diseases, and its conserved sequence would be a new target of drug design.In recent years, many researchers tried to synthesize RGD and RGD-containing peptides by chemical or enzymatic methods. Chemical methods are classical synthesis methods of peptides, which always need short reaction time and high yield. The principle of the enzymatic methods is on the bases of reverse of the hydrolyzation of peptide bonds. As compared to the chemical method, enzymatic peptide synthesis have many important benefits such as mild conditions of the reaction, high regiospecifity of enzyme allowing the use of minimally protected substrates, few of side reactions, small toxicity et al., and chemical method is suitable for synthesis the moderate length peptide and with large-scale. Therefore enzymatic methods have a good applicable foreground. As compared to the chemical method, the important benefits of enzymatic peptide synthesis are:a)the mild conditions of the reaction;b)the high regiospecifity of enzyme allowing the use of minimally protected substrates ; c ) the reaction being stereospecificity without racemization. However, the method also exist in some limit in the employment, the organic solvents must be adopted instead of aqueous solvents for the reverse of peptides hydrolyzation, under this instance, we need to consider the problem that hydrophilic amino acids have low solubility in organic solvents.A number of hydrophobic small peptides were synthesized in high yields using proteases in organic media as largely reported. However,the synthesis of the hydrophilic peptide generally faced some problems:such as substrates solubility,activity and stability of enzyme in organic solvents. In hydrophobic organic solvents,the enzyme usually shows better activity and selectivity,but the solubility of hydrophilic substrates was too low. The polar solvents could increase the solubility of hydrophilic substrates. However,they are often harmful to enzyme because it has a greater tendency to strip the tightly bound essential water from the enzyme molecules. The appropriate reaction system should be selected considering the balance of solubility of substrates and enzymatic activity. RGD tripeptide is composed of three amino acids in which two are hydrophilic and one is neutral amino acid, which make the synthesis of RGD difficult. Consequently, the study of synthesis of RGD in organic solvents has important theory significance and practical application value. So it was selected as the model peptide in this study.In this study, we firstly synthesized free RGD tripeptide by a novel chemical method, not only inheriting the benefits of classical chemical methods but also simple and cheapness. In the first place, we synthesized GD dipeptide using a novel chemical method in two steps including chloroacetylation of L-aspartic acid and ammonolysis of chloroacetylated L-aspartic acid with low cost at large scale. Sencondly the synthesis of RGD was carried out by chemical methods, which include the preparation of NCA-Arg and synthesis of RGD. The methods are benefit to quick reaction rate, shorter reaction time, high yield with low cast at a large scale; reaction amino acid need not to be protected; and the free tripeptide can be adopted to the farther polypeptide synthesis directly. Compared to other method, this method is simple, speediness, cheapness and high yield with about 45%, which is very fit for industrial exploiture.In the next place, owing to the chemical structure characteristic, we succeed in synthesizing the tripeptide RGD ester by combining a novel chemical method with enzymetic method. First of all, free dipeptide, Gly-Asp was synthesized by a novel chemical method only using glycine at a large scale with low cast, followed by esterification of Gly-Asp. The linkage of the third amino acid Bz-Arg-OEt to Gly-Asp-(OEt)2 was completed by enzymatic method in ethanol/ This-HCl buffer system, because ethanol/ This-HCl buffer system is suitable as the reaction medium for the synthesis of hydrophilic amino acid-containing peptides due to better solubility of substrates. RGD tripeptide was in the form of two ethyl ester [Bz-RGD-(OEt)2], which can act as both acyl donor and nucleophile for carrying out peptide chain extension directly without deprotection/activation. The effects of water content, TEA, molar ratio of substrates and reaction time on the yield of Bz-RGD-(OEt)2 synthesis were examined.Recently, it has been reported that RGD can kill human prostate cancer cells (TSU), glomerular mesangial cells from adult human kidneys (GMC) and human leucocythemia cells (HL-60). This study armed to investigate the inhibitory effect of four peptides containing RGD,RGD-(NH22,RGDS,RGDS-NH2 on growth and proliferation of human ileocecal adenocarcinoma cells HCT-8 and induced apoptosis.Firstly, by examining the growth of the human ileocecal adenocarcinoma cells HCT-8 by MTT assay, we found that RGD can inhibit the growth of the cells. We defined the concentration and time of our experiments by adding various concentrations of RGD to the human ileocecal adenocarcinoma cells HCT-8. The final concentration is 50μg/ml and the time is 48 hours.The four RGD-containing peptides RGD、RGD(NH22 (RGE-NH2)、RGDS and RGDS-NH2 had an obvious effect of inhibiton on the growth and proliferation of the cells.Secondly, by observing the morphological of HCT-8 cells,we learned the reason why the RGD-containing celler adhesion peptides, RGD、RGD(NH22 (RGE-NH2)、RGDS and RGDS-NH2 could inhibit the growth of HCT-8 cells. It is because that they could induce the apoptosis of cells. To get more evidence, we observed the apoptosis peak on flowcytometry apparatus, from which we saw that a large amont of cells in multiplication period were blocked in the S period.They could not proliferate and then appeared a heightening in G0/Gl, the obvious decreasing of PI%, and the apoptosis of some cells. The anti-cancer mechanism is probably by reducing the expression of Cyclin Bl and blocking the cells in the period of G2/M. The special DNA ladders could be observed by DNA electrophoresis.To get further information of the moleculer mechanism of apoptosis, which was induced by the RGD-containing peptides, we detected the intracellular anti-apoptosis protein-Survivin by immunohistochemistry experiment. From the contrast to the positive rate and the grey scale value, we knew clearly that the expression of the Survivin protein reduced notably, which also confirmed the apoptosis in HCT-8 cells. We use reverse transcription-polymerase chain reaction (RT-PCR) to detecte the expression of Caspase-3 mRNA. After taking effect on HCT-8 cells by GAPDH、RGD、RGD-(NH22、RGDS and RGDS-NH2, the value of spectroscopical density of mRNA amplification production of Caspase-3 changed, which confirmed that the celler adhesive peptide RGD can induce the apoptosis of HCT-8 cells, and it could be caused by mitochondria. The mechanism is uncertain.we found that cellular adhesion peptide RGDS,RGDS-NH2 ,RGD significantly inhibited the adhesion of HCT-8 cells and the apoptosis in dose-dependent manner. RGD-(NH2)2 did not show obvious ability to inhibit cell adhesion. All of these indicated that RGD tripeptide was a common conservative sequence for cellular adhesion.The results indicated that only the RGD-containing peptides(RGD,RGDS,RGDS-NH2) could induce the apoptosis of HCT-8 cells. It is probably because that the celler adhesive peptides could effect on tumor cells by the the conservative sequence of RGD, and the effect of RGDS and RGDS-NH2 is much stronger than RGD. Actually the primary structure of RGD-(NH22 should be RGE-NH2 , which did not contain RGD. Therefore it has neither the target effect, nor the effect of inducing cell apoptosis. It could be identitied by the reports before. As a result, we confirmed that RGD was high-conservative in mediating cell apoptosis.It was reported that tumor was caused by two reasons, one is the overexpression of oncogenes which controlled the cell proliferation, and the other is the high expression of oncogenes which inhibited cell apoptosis and the devitalizate and variation of the antioncogene.Recently, though one takes much attention on entering cells directly by RGD to induce celler apoptosis, the research on RGD inducing tumor cells apoptosis is still not much. The effect on RGD inducing the human ileocecal adenocarcinoma cells HCT-8 in our experiment made much sense of tumor prevention and cure, and made it may become the theoretic base in the application of the tumor therapy.The reaction steps of RGD synthesis is:

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