A Preliminary Study On The Killing Activity And Molecular Mechanism Of Acylated CM4 With Different Chain Lengths On Breast Cancer Cells

Cancer is a threat to human life and health,one of the most important disease in all kinds of disease,the mortality rate is second only to disease of heart head blood-vessel,ranks second.Chemotherapy,as one of the main means of removing cancer cells,plays a great role in curing cancer cells,but because of its itself has a lot of side effect and low degree of differentiation of normal cells and cancer cells,and is also easy to cause tumor cell resistance,makes its clinical application limited.So to hunt for a new anti-cancer resources with high specificity,high power and low toxicity to normal cells become a research hotspot.Antibacterial peptide is a kind of widely exists in the nature of cationic peptides,antimicrobial peptides have a broad antibacterial spectrum.In addition to possess the ability of antibacterial acivity,Antibacterial peptides also possese the ability of antuviral activity,antifungals activity,antiprotozoa activity and anticancer activity.Up to now,about 180 species of antibacterial peptides have been isolated,makeing the antimicrobial peptides become a new kind of anticancer resources.However natural antibacterial peptides is limited by anticancer activity and selectivity,it can’t directly as anti-cancer drugs.Antibacterial peptide CM4 is a cationic antimicrobial peptides composed of 36 amino acids,has a certain anti-cancer activity,but the antitumor activity is not high and anticancer spectrum is narrow,however,its high selectivity had made it be a potential anticancer drug.For enhancing the antitumor activity of antibacterial peptide CM4 without changing its selective become an urgent need to research topic.In order to solve the problems,many scholars try to modificate it wuth chemical method,including heparin,polysaccharide and modified polyethylene glycol,these modifications to a certain extent,improve the stability of antibacterial peptides,extended the half-life of antimicrobial peptides,but this chemical modifications increase the hydrophilia and the relative molecular mass of this peptide,and lower the ability to target cell membrane.Fatty acid modification can improve the stability and hydrophobic of this peptide,making it more easier to combine with membrane.Therefore our experiment use butyric acid,caproic acid,octanoic acid t,Deca-Durabolin,lauric acid,myristic acid,palmitic acid to modificate antibacterial peptide CM4,to explore the fat acylating modified antitumor activity of antimicrobial peptides in regulation and mechanism,which provides the theoretical foundation for clinical application.1,Antibacterial peptide CM4 was modified to synthesis Butyl acylation CM4(But-CM4),Caproic acylation CM4(Cap-CM4),Octanoic acylation CM4(Oct-CM4),Deca-Durabolin acylation CM4(Dec-CM4),Lauric acylation CM4(lau-CM4),Myristic acylation CM4(Myr-CM4),Palmitic acylation CM4(Pal-CM4).2,MTT experiment tests the antibacterial peptide CM4,Butyl acylation CM4(But-CM4),Caproic acylation CM4(Cap-CM4),Octanoic acylation CM4(Oct-CM4),Deca-Durabolin acylation CM4(Dec-CM4),Lauric acylation CM4(lau-CM4),Myristic acylation CM4(Myr-CM4),Palmitic acylation CM4(Pal-CM4)on breast cancer cell(MCF-7,MX-1),prostate cancer cells(PC-3,DU-145),non-hodgkin’s lymphoma cells(Raji,Daudi),lung cancer cells(A549)seven the killing activity of tumor cells,the results show that several kinds of fat acylation modification can significantly improve the CM4 on a wide variety of tumor cell killing activity.Compared with the undecorated CM4,IC50(half inhibitory concentration)reduces the 25 to 35 times,approximately 3-6μM.3,MTT experiment tests the antibacterial peptide CM4,Butyl acylation CM4(But-CM4),Caproic acylation CM4(Cap-CM4),Octanoic acylation CM4(Oct-CM4),Deca-Durabolin acylation CM4(Dec-CM4),Lauric acylation CM4(lau-CM4),Myristic acylation CM4(Myr-CM4),Palmitic acylation CM4(Pal-CM4)on the three kinds of normal mouse embryonic fibroblast cells(NIH3T3),human normal liver cell(LO2),human normal prostate stromal immortalized cells(wpmy-1)toxicity,in concentrations reach 40μM,the three normal cell survival rate reached more than 75%,far less than the toxicity of cancer cells,showing obvious selectivity.Acyl-CM4 also strengthened the toxicity to normal cells,hemolytic experiment was carried out on the kunming eye red blood cells in mice,the results show that fat acylating modified antibacterial peptide CM4 can improve the hemolytic,and the longer the fatty acyl chain,hemolytic,the more obvious,but within the scope of the use don’t appear hemolysis.CD spectroscopy fatty acyl modification on secondary structure,the influence of CM4 results show that several kinds of fatty acyl can improve the CM4 alpha helix structure content,fatty acid,the longer the chain of alpha helix structure content increase,the more obvious,fatty acid chain without change the secondary structure of CM4 species.Fatty acids,therefore,can significantly improve the antibacterial peptide CM4 killing activity of tumor cells,and does not change the CM4 selectivity.4,Fluorescent tags streaming cell analysis show the results that several kinds of fatty acids of CM4 significantly enhanced and MCF-7 cell of breast cancer combined with ability,but also enhances the ability of the combination of normal cell NIH3T3,but combining ability of the breast cancer cells is greater than the binding ability of normal cells.Laser confocal fat acylation CM4 and MCF-7 cell and the combination of NIH3T3 cells,the results show that butyl acylation and acylation enhances the CM4 and MCF-7 membrane combining ability,lauroyl and palmitoyl mediated the CM4 across the plasma membrane,and the longer the chain fatty acids,across the plasma membrane.5,The results of LDH release experiment show that under the low concentration acyl-CM4 for MCF-7 cell membrane damage is not apparent,high concentrations fat acylation CM4 certainly more MCF-7 cell membrane damage,has a long time and fatty acyl chain dependencies.DAPI staining and AnnexinV/PI double dyeing experiments show that several kinds of fat acylating modified in the form of apoptosis inducing CM4 MCF-7 deaths,with the increase of concentration and fatty acyl chain increases,the apoptosis rate increased.PI single dye showed that fat acylation CM4 MCF-7 cell cycle arrest,mainly in S phase.6,The results of ROS experiment show that fat acylation CM4 rise the content of MCF-7 cell ROS,acyl-CM4 can destroy the function of mitochondria,and lead to the decrease of mitochondrial membrane potential.Immune imprinting experiments show that acyl-CM4 induced apoptosis related proteins PARP,Caspase-3,Caspase-9 cleaved,bax expression rise,the Bcl-2 expression decline,cycle related protein expression CyclinA drops,CyclinDl has no obvious change,CyclinE1 expression elevated.Results show that acyl-CM4 can induced apoptosis of breast cancerthrough the mitochondrial pathway.The study was carried out by different chain length fatty acyl modification of CM4 antitumor activity and molecular mechanism of the effect of preliminary studies,suggest that fatty acyl modification can significantly enhance the CM4 anticancer activity,and the longer the chain length,the stronger the antitumor activity,but also enhanced the toxicity to normal cells and hemolysis activity.Fatty acyl promoted the combination of antibacterial peptide CM4 and cell surface ability and the ability across the plasma membrane,can through the change of mitochondrial function,induction of reactive oxygen species generation,breast cancer cells induced apoptosis of MCF-7.This research results to design high anticancer activity,low toxic side effects of lipid acylating modified antibacterial peptide has important theoretical value.