Study On The Modification Of Lycosin-? And Its Bioactivities

Cancer is one of the serious diseases which threatens human health and even life.The efficient treatment of cancer has become research hotspot in the field of medicine.Conventional anticancer peptides get extensive application in experimental research for their outstanding characteristics of highly selective targeting,high activity,easy permeation and absorption.However,their clinical application is limited by several shortcomings such as short half-time and degradation of protease easily.Therefore,it is of great significance to study the structural modification of ACPs to resolve the deficiency of ACPs.In this study,Lycosin-? is a linear amphipathic anticancer peptide,which extracted from the spider Lycosa singoriensis.Amino acid substitution strategy was used to replace the lysine in the Lycosin-? sequence with arginine to obtain Lycosin-? similarity,which named R-Lycosin-?.The physicochemical structure of R-lycosin-? was characterized by DLS,CD and TEM,and studied on its activity,serum stability and mechanism of action.The DLS results indicated that decreased in particle size and increased in zeta potential.Circular dichroism spectroscopy results showed that both Lycosin-? and R-lycosin-? showed random curls in PBS solution and exhibited a-helical structure in TFE solution.TEM showed that the morphology of Lycosin-? and R-lycosin-? were spherical.The anticancer activity results indicated that R-lycosin-? improved the anticancer activity nearly 2 times as well as increased the selectivity for cancer cells over non-cancer cells.Cellular uptake and release studies have shown that R-lycosin-? is more readily taken up and taken up faster by A549 cells than Lycosin-?.After ingestion into the cells,the modified R-lycosin-? was more easily released and showed a diffuse state in the cells,while Lycosin-? showed a dotted distribution.The results of serum stability study showed that the inhibition rate of R-lycosin-? on A549 cells was 80%at 8 hours,while Lycosin-? activity had lost about 50%at 30 minutes.By simulating 3D tumor experiments in vivo,the results confirmed that R-lycosin-? has a stronger inhibitory effect on tumor spheres.The penetration ability of R-lycosin-? in solid tumor spheres was studied by Laser confocal Z-scanning.The results showed that R-lycosin-? has stronger tumor penetration,which penetrates the tumor sphere about 30 ?m,while Lycosin-? is only 10 ?m.In the Western Blot study,R-lycosin-? was found to induce apoptosis by upregulating the expression of P27 to inhibit cell proliferation and promote the release of cytochrome C.In order to further improve the activity and selectivity of R-lycosin-?,it is proposed to further modify R-lycosin-? by adopting a glycosylation modification strategy.Five kinds of monosaccharide derivatives such as glucose,galactose,arabinose,mannose,glucosamine were used as modifying units,glutaric anhydride was used as a linker,and glycosylation of R-lycosin-? was performed to obtain five glycopeptides.The physicochemical structure of glycosylated R-lycosin-? was characterized by DLS,TEM and CD,and studied on its bioactivities.DLS showed that the zeta potentials of the five glycopeptides were between 5.0±0.35 mV and 58.9±0.35 mV,and the mean particle sizes were between 189.0±0.47 nm and 79.3±1.3 nm.TEM showed that the morphology of five glycopeptides were spherical.The results of CD spectra showed that all the five glycopeptides showed random curls structure in PBS solution and showed a-helices structure appeared in TFE solution.The results of CCK-8 activity experiments showed that different monosaccharide derivatives modified R-lycosin-? activity were not the same.Compared to the anticancer activity of R-lycosin-? on A549 cells,the anticancer activity of Glu-R-lycosin-? increased nearly 1.8 times;the anticancer activity of Gal-R-lycosion-I increased about 2.5 times;Man-R-lycosin-? activity is almost lost(?100 ?M);Ara-R-lycosin-? and GluNAc the anticancer activity of-R-lycosin-? are almost unchanged.The results of serum stability experiments showed that the inhibition rate of Gal-R-lycosion-I and Glu-R-lycosin-? was still 80%at 48 hours.Serum stability is greatly enhanced.In summary,relative to Lycosin-?,R-lycosin-? obtained by site-directed amino acid mutations significantly enhanced the biological activity such as the stability of the serum and the activities of resistance to solid tumors.Through the glycosylation of R-lycosin-?,it was found that different glycosyl units have different effects on the activity of glycopeptides.This finding provides a basis for the subsequent analysis of the mechanism of action of glycopeptides,enabling to have further study on the value of the development of Lycosin-?.