Study On The Application Of Lipidated Antimicrobial Peptide Camel In Tumor Therapy

With increasing incidence and mortality,cancer is the leading cause of death in the world,which seriously threatens public healthy.Therapy depending on cytotoxic agents remains the major method for cancer treatment.However,severe side effects due to the lack of selectivity for cancer cells over normal cells and occurrence of drug resistance are the major drawbacks of traditional cytotoxic agents.Gene therapy has become one of the preferred methods for the treatment of some tumor.However,the naked nucleic acids are unable to translocate across the cell membrane.Thus,it is very critical to develope evectors that can efficiently deliver nucleic acid drugs into cells.Recently,antimicrobial peptides(AMPs)have received increasing attention as potential candidates for anticancer drugs because their target is cell membranes rather than specific receptors.Based on the unique mechanism of action involving membrane-disruption,antimicrobial peptides display acertain degree of selectivity to cancer cells and disfavor the development of drug-resistance compared with traditional cytotoxic agents.It is reported that some antibacterial peptides could effectively ranslocate across the cell membrane.So,these antibacterial peptides also used applicated as the cell penetrating peptides.Stearylation has been proven to be a successful methodology to increase the transfection efficiency of cell penetrating peptides.Thus we have developed stearyl-melittin and stearyl-K6L9.Their transfection efficiency almost reached the transfection levels of Lipofectamine 2000.Camel,a synthetic hybrid peptide,made up from cecropin A and melittin with strong membrane-lytic activity,has been used as an anticancer peptide.Therefore,in this study,we introduced the stearylation moiety to the N-terminus of antimicrobial peptide Camel to improve their anticancer activity and cell penetrating activity.Our research results showed that the Camel and lipidated Camel has excellent anticancer activity.Short fatty acids analogues of 4-Camel and 8-Camel showed rapid membrane-lytic activity to cancer cell membranes,so they have fast antitumor activity.While long fatty acids analogue of 16-Camel and 18-Camel lost the rapid membrane-lytic activity,but after the long time 72 h they showed stronger antitumor activity.After cellular internalization,16-Camel and 18-Camel localized to mitochondria and lowered the mitochondrial potential,resulting in the organelles' swelling,a decrease in cellular ATP level and,finally,cellular breakdown.Other research results showed that the transfection efficiency of 12-Camel begain increased and 16-Camel as well as 18-Camel showed obvious increases in transfection efficiency compared with Camel.Moreover the transfection efficiency of 18-Camel almost reached the transfection levels of Lipofectamine 2000.We also find that 18-Camel delivered plasmids into cells by clathrin-and caveolin-mediated endocytosis.Our datas also indicating that 18-Camel could be used as an efficient endosome-lytic agent to enhance gene transfer.In addition,the presence of serum only had a modest effect on the transfection efficiency of stearyl-Camel.Furthermore,the 18-camel/p53 plasmid complex exhibited high p53 expression and antitumor activity in MCF-7,MDA-MB-231,U87-MG and U251 cell;at the same time,stearyl-camel/survivin siRNA complex exhibited high antitumor activity to MCF-7 and MDA-MB-231 cell by silence survivin expression.Taken together,we developed a highly efficient anti-cancer peptide,at the same time,it is a highly efficient nonviral gene vectors,and provide a excellent nonviral gene vectors for the tumor gene therapy.