Study On The Biological Function Of The Antimicrobial Peptide Scolopin-2 And Its Amidation Modification

Currently,cancer has become the major disease affecting human health.To find efficiently anticancer drugs become the main direction of cancer therapy.Antimicrobial peptides(antimicrobial peptides)are the important part of innate immune system in a variety of biological body.What’s more,they have no toxicity to normal eukaryotic cells and have a broad spectrum of antibacterial activity.In addition to the familiar anti-bacterial and ingal activity,they also have anti-viral,anti-cancer and other functions.More importantly,due to the overuse of antibiotics leads to resistant strains present,and the unique antibacterial mechanism of the antimicrobial peptides,it has become desirable to develop a new class of broad-spectrum antimicrobial,anticancer drugs.Scolopin-2 was isolated from centipede venoms of Scolopendra subspinipes mutilans.It is a linear and a typical amphipathic α-helix cationic peptides by 25 amino acids with a wider antibacterial,antiviral and anti-ability of some tumor cells.However,compared with traditional antibiotics and anti-tumor drugs,the antibacterial activity and anti-tumor effects of antimicrobial peptides Scolopin-2 are still not ideal,it works only at high concentrations.In order to find a ideal antimicrobial peptide,this study went into molecular modification of the Scolopin-2.Our research is divided into three parts:1.To improve the antimicrobial peptides of bacterial,anti-tumor effect,we designed the following three kinds of mutants:The C-terminal histidine amidated:Scolopin-2-NH2,sequence:GILKKFMLHRGTKVYKMRTLSKRSH-NH2;N side plus RGD:RGD-Scolopin-2,sequence:RGDGILKKFMLHRGTKVYKMRTLSKRSH;N terminal plus the RGD sequence and the C-terminal histidine amidated:RGD-Scolopin-2-NH2,sequence:RGDGILKKFMLHRGTKVYKMRTLSKRSH-NH2The agarose cavity method was used to detect the killing of four antimicrobial peptides on Escherichia coli and Staphylococcus aureus.The results show that Scolopin-2 and amidation mutant have obviously antibacterial effect.Methyl thiazolyl tetrazolium(MTT)assay showed that the amide Scolopin-2 mutant could inhibit proliferation of tumor cells in a dose-dependent manner,and the effect of amidation Scolopin-2 was significantly stronger than Scolopin-2.So we choose amidated Scolopin-2 to study the anti-tumor mechanism.2.Flow cytometry was used to assess the apoptosis.Results demonstrated that both antimicrobial Scolopin-2 and amidation Scolopin-2 can induce HepG2,Hela and K562 apoptosis.Confocal laser scanning microscopy assay indicated that amidation Scolopin-2 can pass cytomembrane and location on the membrane of mitochondria of HepG2,Hela and K562 cells.The results showed that the amidation Scolopin-2 can enhance the ability to induce apoptosis of cervical cancer Hela cells,HepG2 cells and K562 cells,and they combined with the cell mitochondria,indicating amidated Scolopin-2 induces apoptosis by the way killing tumor cells and prompts the mechanism associated with the mitochondrial pathway.3.Changes of apoptosis-related proteins level were detected by western blot.The results showed that Scolopin-2 and amidation Scolopin-2 can change the proteins of tumor cells.After treated with the two peptides the expression level of caspase-3,PARP and Bcl-2 decline and the expression level of Bax increase in Hela cells,HepG2 cells and K562 cells.The results indicate that the two peptides can induce tumor cell apoptosis via the mitochondrial pathway.4.In vivo level,we constructed the transplanted tumor model of Hela cervical carcinoma in nude mice successfully.High and low doses of Scolopin-2-NH2 could inhibit the growth of cervical cancer xenografts in nude mice.In vivo,immunochemical staining of tissue sections showed Scolopin-2-NH2 can inhibit tumor cell proliferation and inhibition of angiogenesis to inhibit tumor growth.In summary,after the C-terminal amidated the antimicrobial peptide Scolopin-2 has stronger activity on inducing tumor cell apoptosis,the apoptosis mechanism associated with the mitochondrial pathway and it can significantly inhibit cervical cancer xenografts in nude mice.These results would provide experimental and theoretical basis for the clinical treatment of cancer.