Research Of Anticancer Peptide From Meretrix Meretrix Linnaeus

Cancer is one of the most dangerous diseases of human being. In nowadays, it has the highest mortality in place of blood vessel disease in china and is worthy of the name of "the first killer". Many researches were reported that the anticancer component from natural organism could combine with special regulation target during the course of growth, develop and malignance of cancer. Screening out those active substances have been the hotspot in the work of finding new effective anticancer medicament, and it is also one of the most important aspects in research of biomedicine in china now. Traditional anticancer medicines have many disadvantages such as heavy side-effect and toxicity, nonspeciality, facility of antigen and so on. Compared with them, the anticancer peptides from natural organism have many excellences, including multifunction, sensitivity, stability, lower side-effect and lower toxicity, and it has no antigen character also. Furthermore, the synthesis of the anticancer peptide is ease because of its simple structure. Meretrix Meretrix Linnaeus are smooth-shelled marine mussels.They are delicious and have medicinal properties. Many researchers have reported that extraction from Mercenenia had inhibitory effect on cancer of lung, gastrintestine, liver and hypothyroid, and there also were plenty of researches about its function of depressing blood sugar and lipid. It was also reported that extraction from Mercenenia had resistance to decrepitude, oxidation and nucleus mutation. Furthermore, some people reported that there was powerful immunocompetence in extraction from Mercenenia. Now more and more researchers are intresting in study of anticancer active of extract from Meretrix meretrix Linnaeus.In this study, in order to purify the active component from Mercenenia, the purification protocol was optimized at first. Then, on this base, we used a series of methods including fragmentation, organic precipitation, column chromatogram and HPLC. Finally, the anticancer peptide was obtained. The peptide was determined to be single band on the gel of PAGE, and we named it as MP1.The research of cell biology indicated that MP1 could strongly inhibited hepatocarcinoma cells SMMC-7721, gastrintestinal tumour cells SGC-7901 and BGC-823 inlow concentration, and the IC₅₀ to those cells were all lower than 5μg/mL.The inhibitory effects of MP1 on the SMMC-7721 cells, including its effects on their proliferation, microstructure, ultrastructure and cell-cycle, were first been investigated. The cell proliferation and the inhibition rates were determined by cell counting methods and MTT methods, respectively. The microstructures and ultrastructure of the cells were observed by scan electron microscope and transmission electron microscope, respectively. The cell cycle was analyzed by flow cytometry. It was found that the SMMC-7721 cells, after they were treated by 5.0μg/mL of MP1, grew slower than the control, their doubling time were postponed and the inhibitory rate on the cell growth was determined to be 89.4%. In the mean time, flow cytometry analysis showed that the cell proportions in G₀/G₁ and M phases were significantly decreased and what in the S phase was increased markedly. The apoptosis rate was up to 9.29%. Electron microscope results revealed that the cells' morphological, microstructural and ultrastructural changes exhibited obviousl apoptotsis properties. These results indicated that MP1 could effectively inhibit the proliferation of the human hepatocarcinoma cell line SMMC-7721 in vitro and might act as an apoptosis inducer in its antitumor effects via inducing morphological changes and cell cycle arrest.Proteomics technology was used to study the potential molecular mechanism of inhibitory effects of MP1 on the SMMC-7721. 2-D patterns were obtained and analysised by using 2-D software-melanie 4. Proteins were digested by enzyme trypsin in gel and MALDI-TOF analysis were carried out in the proper order. Finally, we identified differential proteins expressed in the gels by searching protein database.About 136 differential expressed proteins were found on two patterns. One was the control cells proteins pattern, the other was that of cells treated by MP1. There were 96 down-regulated proteins and 40 up-regulated proteins in two pieces of patterns. We choosed some proteins which changed markedly and identified them by MS. 11 proteins were identified successfully, 6 down-regulated proteins and 5 up-regulated proteins. These proteins included cyskeletal protein, zinc finger protein, metabolism-related enzyme, transcription regulation factors and so on. Finally, we discussed the fuctions of some of these proteins and attempted to come up with the hypotheses to explain the potential molecular mechanism of inhibitory effects of MP1 on the SMMC-7721.