The Reaction Of Cell Membrane Between Phospholipids And Temporin-1CEa,Its Analogues

Antimicrobial peptides(AMPs),as potential anti-cancer drugs,have been widely studied because of its strong cancer selectivity,less side effects and less resistance.In this paper,Temporin-1CEa,a natural cationic AMP,as well as its peptidic analogues LK1,LK2(6)and LK2(6)A(L)were selected.Their net positive charge is +3,+4,+6,+6.Our preliminary study found that these peptides exerted good cationic,hydrophobic,hydrophilic and typical alpha helix structure,and could selectively interact with cancer cells membrane without cytotoxicity to normal cells.The membrane phospholipid composition is generally considered to be the main biological basis for the different response to AMPs between cancer cells and normal cells.In the present study,we will discuss the interactions of Temporin-1CEa and its analogues with membrane phospholipids,and then find out the cellular mechanisms for the cationic AMPs selective effects on cancer cells.Moreover,the structure-activity relationship will be further analyzed.First,we investigated the membrane charge variation of five breast cancer cell lines after being exposed to AMPs,by using Zeta detection.Our data showed that the more positive charge AMPs carrying,the more membrane negative charge is neutralized.The ELISA-Like assay showed that the biotin or fluorescein-labeled AMPs could interact directly with negatively charged phospholipids PS,PA,PI and PG,but not with neutral phospholipid PE,PC and SM.We then determined the interactions between AMPs with membrane phospholipids by higher sensitive Lipid dot blot assay.The results further found that AMPs showed better interaction with PS.Additionally,Temporin-1CEa and LK1 also interacted with PA,LK2(6)and LK2(6)A(L)interacted with PI.The results suggested that AMPs combined easily with negatively charged phospholipids,with PS was the best.The degree of binding was LK2(6)A(L)> LK2(6)> LK1 > Temporin-1CEa.Because PS is the most abundant negatively charged phospholipids on cancer cells surface,and four kinds of AMPs all showed strong binding with PS,therefore,we choose PS as the targeting molecule to further detect the molecular interactions between peptides and phospholipids.Based on the above experiments,we tested four kinds of AMPs and PS affinity by surface plasmon resonance.The results indicated that four kinds of AMPs showed much higher affinity to PS than neutral PC.The affinity sequence is LK2(6)A(L)> LK2(6)> LK1 > Temporin-1CEa.Next,we determined the potential inpacts of PS on peptides secondary structure by CD spectrum.The results showed that AMPs displayed irregular curling in the SP buffer,whereas in the phospholipid-containing environment,four kinds of AMPs were induced to form alpha helix structure under a PS-related manner.The results suggested that PS not only was the binding targets for Temporin-1CEa and its analogues,but also induced more alpha helix structure of AMPs,thereafter to play anti-cancer activities.In summary,Temporin-1CEa and its peptidic analogues selectively combined with negatively charged phospholipids,with PS strongest affinity.PS can promote the formation of alpha helix structure.All these findings provided experimental evidence that the negatively charged phospholipids such as PS play important roles for the selective interaction and cytotoxicity of AMPs to cancer cells.