The Research Of Novel Polypeptide MM-16 From Meretrix Meretrix Linnaeus And Its Anti-tumor Activity

Today,malignant tumors have become one of the principal diseases that threaten human life and health.Methods commonly used to treat malignant tumors are excessively toxic and have side effects,such as radiotherapy and chemotherapy,etc.And we urgently need to invest more social resources to find and discover new antineoplastic drugs and methods that have good curative effects and low side effects.In recent decades,with the development of biology and related disciplines,a variety of natural bioactive substances have been gradually discovered and applied to the human production and life.And they are playing an increasingly important role in chemical production,health care,prevention and control of pollution,and other fields.After a large number of literature search and screening experiments,we found that Meretrix meretrix Linnaeus,a marine organism,contains a variety of bioactive substances,including anti-tumor activity.Therefore,this research aims at exploring a set of separation and purification process to obtain a new polypeptide from the clam,which has anti-tumor cativity and studying its biological activity.A novel anti-tumor polypeptide was extracted and purified from the coelomic fluid of Meretrix meretrix Linnaeus by ammonium sulphate precipitation,CM-Sepharose Fast Flow weak cation exchange chromatography,Phenyl Sepharose 6Fast Flow hydrophobic interaction chromatography,Superdex 75 prep grade gel filtration and Q Sepharose Fast Flow strong antion exchange chromatography.And we detected the molecular weight and the proliferation inhibitory activity of tumor cells in vitro of the polypeptide from Meretrix meretrix Linnaeus.SDS-polyacrylamide gel electrophoresis?SDS-PAGE?detection showed a single band with a molecular mass of about 16 KDa and this peptide was designated MM-16.The effect of polypeptide MM-16 on viability of tumor cell lines as well as benign cell lines was assessed by MTT assay.Results showed that the half inhibitory concentrations(IC₅₀)of MM-16 on human colorectal cancer cell line HCT-116,human ovarian carcinoma cell line HeLa,human hepatoma cell line BEL-7402,human lung cancer cell line A549,human pancreatic cell line Aspc-1,normal mammary gland cell line MCF-10A and mouse embryonic fibroblasts NIH/3T3 are 65.50,42.88,51.86,33.68,56.68,196.86 and 147.67?g/m L,respectively.Therefore,we can draw a conclusion that polypeptide MM-16 inhibites the growth of five kinds of tumor cells and the proliferation inhibitory rate of human lung cancer cell line A549 is the highest in vitro.However,MM-16 did not significantly decrease the viability of two benign cells.Besides,the influences of dosing concentration and relation time of MM-16 were assessed on human lung cancer cell line A549.And the result showed that MM-16 has a dose-effect relationship on the proliferation inhibitory activity of A549 cell lines.The inhibitory effect reached the maximum after 48h and continued to more than 72h.The separation and purification and the bioactivity research of anti-tumor polypeptide MM-16 has laid a foundation for its future anti-tumor mechanism research,its establish of recombinant expression technology and its mass production.Our study provides reference for the development and expansion of antitumor drug field and prevention of malignant tumor.Consequently,it is of great significance.