Antitumor Mechanisms Of Small Molecular Peptides Revealed By Atomic Force Microscopy

Cancer has become a major problem endangering human life at present.Compared with traditional cancer treatment methods,antitumor peptides have the advantages of high antitumor activity,low cytotoxicity,and less resistance to drug resistance,which has become a new focus of anticancer drug research now.According to the structural characteristics of natural antitumor peptides,we have designed two amphiphilic small molecule polypeptides G（IIKK）₃I-NH₂（G3）and（IIKK）₃-NH₂（X）,and it was found that although both of them areα-helical peptides,they have different anti-tumor activities.In order to study the anti-tumor mechanism of these two peptide and structure-activity influence on its anti-tumor effect at the molecular level,this research explored the method to test how small molecular anti-tumor polypeptides treat cells by atomic force microscope（AFM）,and studied the change of SY5Y cells morphology and mechanical properties treated with the two kinds of small molecular peptides,with the advantage of AFM biological sample imaging.It was found that G（IIKK）₃I-NH₂ has obvious stages in the antitumor process,and has strong concentration dependence.According to the changes of cell morphology and mechanical information when SY5Y cells are treated at IC₉₀concentration,the action process of IC₉₀ is divided into three stages.The first stage was the stage of molecular membrane insertion.The morphology of cells shows no significant difference,the height decreases slightly,the Young’s modulus increases slightly,and the adhesion force increases.The second stage is the molecular transmembrane stage,the surface roughness and height of cells decrease,and Young’s modulus and adhesion force decrease.The third stage is the membrane breaking stage,with obvious cytoplasmic leakage,Young’s modulus is no longer normally distributed,and surface roughness increases.In a short period of time,the mode of G（IIKK）₃I-NH₂ is highly dependent on the concentration.At low concentrations(1～2×IC₉₀),the main function of G（IIKK）₃I-NH₂ was membrane insertion and cell membrane disturbance.However,at the medium concentration(5×IC₉₀),G（IIKK）₃I-NH₂ combines with the membrane and reaches saturation,and can cross the membrane into the cell.At higher concentrations(>10×IC₉₀),G（IIKK）₃I-NH₂ can quickly cross the membrane and destroy the cell membrane and internal structure.Compared with G（IIKK）₃I-NH₂ at the concentration of IC₉₀,the tumor cells were killed by"membrane insertion followed by transmembrane at last membrane rupture",after the treation of（IIKK）₃-NH₂ with the same concentration,the molecules could not cross the membrane to enter the cell interior and destroy the cell membrane structure after the insertion reached saturation,but could only aggregate the cell surface on the surface of the cell membrane.Therefore,the roughness of the cell would increase,and Young’s modulus fluctuates,but eventually it returns to the initial range.In this study,AFM imaging and force curve measurement were combined to reveal the antitumor mechanism of small molecule peptides,and this study provides certain experimental basis and theoretical basis for the future design and synthesis of novel antitumor small molecule peptides with high efficiency and low toxicity.