Chemo-enzymatic Syntheses Of Cellular Adhesion Motif RGD(S) And Prelimenary Studies On Effects Of Gastric-tumor Cell BGC-823 And Inducing Its Apoptosis

Many proteins present in extra cellar 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-containing peptides as competitive,reversible inhibitors for the binding of adhesive proteins have been widely used to study adhesive interaction between cells and inhibit tumor metastasis,tumor induced angiogenesis and tumor elicited platelet aggregations. RGD peptides not only play a major role as anchoring molecules but also are important in processes like embryogenesis,cell differentiation,immune-response. X1-RGD-X2(X1 =Gly,Asp;X2 =Ser,Cys,Val,Phe),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 peptide exhibits additional potent anti-chemotactic and pro-apoptotic effects independently from its anti-adhesive action,likely by entering the cells and directly activating caspase 8 and 9,and lately caspase 3,implying unexpected intracellular actions of the RGD-motif. In addition,it was also reported that the activity of RGDS is improved upon acetylation of the N-terminus at arginine and amidation of serine at the C-terminus(Ac-RGDS-NH2).As we know,it is not available to isolate RGD or other biologically active peptides from whole tissue directly. So many researchers tried to synthesize RGD and RGD-containing peptides by chemical or enzymatic methods. Chemical and enzymatic method has different characteristic and suit different synthesis scale. Chemical method is suitable for synthesis the moderate length peptide and with large-scale. An enzymatic method is suitable for synthesis the short peptide and with small-scale. 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.For enzymatic method,a number of proteases have been used as practical catalysts in peptide synthesis,particularly in synthesis of small peptides. For protease-catalyzed synthesis of peptides under the kinetic control in organic solvents,the enzymes are limited to serine and cysteine proteases which could form acyl-enzyme intermediate with the acyl donor. Acyl transfer reaction is completed by deacylation of the covalent acyl-enzyme intermediate via possible two pathways of aminolysis and hydrolysis.Although protease-catalyzed synthesis of peptides has several advantages over chemical methods,such as no racemization,no essential requirement of side chain protection,and good stereo- and regioselectivity,but its shortcomings including an undesirable hydrolysis of the growing peptide chain due to an amidase activity,a narrow substrate specificity,and deactivation due to organic solvents still hamper its use in peptide synthesis with long sequence.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 to 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(S)is a good hydrophilic peptide model containing three charged or polar residues (Arg,Asp and Ser)and a neutral one(Gly). So it was selected as the model peptide in this study.In this study,we succeed in synthesizing the tripeptide Bz-RGD(-NH2)2 and the tetrapeptide Bz-RGDS-NH2 by combining a novel chemical method with enzyme method successfully. First,the chemical method was used to prepare the tripeptide GDS-NH2 at large scale with low cost. The linkage of the Arg was completed by enzymatic method under kinetic control in organic media. Trypsin was selected to the synthesis of the peptide bond with Bz-Arg-OEt as the acyl donor. The synthesis reaction conditions were optimized by examining the effects of several factors including water content,temperature, pH,and reaction time on the yields of target peptides. Trypsin was selected due to its substrate specificity suitable for the synthesis of the peptide bond with Bz-Arg-OEt as the acyl donor. In addition,when the trypsin pre-treated with Tris-HCl was used,we found that the yield of the target peptide was increased markedly. Ethanol as the solvent has many advantages,for instance,no toxicity problem and very low boiling point, therefore easily to be removed from the reaction system. The optimum conditions for Bz-RGD-(NH2)2 were pH 8.0,30℃,in ethanol/ Tris-HCl buffer system (85:15,V/V) for 80 min in the maximum yield of 74.4% with pre-treated trypsin and 61.9% with trypsin powder. The optimum conditions for Bz-RGDS-NH2 were pH 8.0,30℃,in ethanol/This-HCl buffer system (97/3,V/ V)for 14h in the maximum yield of 68.3±1.74% with pre-treated trypsin and 61.2% with trypsin powder.. Compared with trypsin powder,pre-treated trypsin possesses higher catalytic activity and stability. Accidentally,in low-water organic media for synthesis Bz-RGDS-NH2,the secondary hydrolysis of the tetrapeptide product did not take place. But for synthesis Bz-RGD-(NH2)2 in ethanol/ Tris-HCl buffer system(85:15,V/V),when the reaction time was over 90min, the yield of the tripeptide product decreased rapidly due to the obvious hydrolysis of the tripeptide product.This study armed to investigate the inhibitory effect of RGDS on growth and proliferation of human ileocecal adenocarcinoma cells 823 and induced apoptosis. We test the proliferation of 823 cells by the MTT assay and observe cell morphological feature and ultramicroscopic structure of apoptotic cells. We find RGDS can inhibit proliferation of 823 cells by induce it apoptosis. For approve it further, flow cytometry analysis and electrophoresis of DNA are done to test apoptosis. we can find a apoptotic peak. With the concentration of RGD increasing, proportion of apoptotic peak increased. In figure of electrophoresis of DNA, there is a typical DNA ladder of apoptotic cells. These results indicate that RGDS do induce apoptosis of 823 cells and the RGDS concentration of 50μg/ml is the best condition. In addition, we display anti-apoptosis protein survivin in cells by way of immunohistochemistry staining and find that RGDS can inhibit expression of Survivin. It suggests that it may be a mechanism of RGDS inducing apoptosis. We would do more study to approve this point later.Nowadays, it has attracted many attentions that RGDS can directly enter into cells and induce apoptosis. But study on this field is still not enough. So our study can base the theory of cure of tumor by RGDS.