Comparison And Analysis Of Four Cytotoxic Peptides From The Venom Of Spider Lycosa Singoriensis

Lycosa singoriensis is one of most poisonous spiders.It mainly distributed in Xinjiang、Gans、Neimenggu and Jilin province of china. The crude venom of Lycosa singoriensis contains a mixture of compounds that can inhibit bacterial growth and progenitive. Trough our deeper research to lycosa singoriensis venom,we found four cytotoxic peptide of anticancer activity. Named lycosinⅠ,lycosinⅡ,lycosinⅢ and lycosinIV,which have 23,22,21 and 19 amino acid residues respectively.And these four peptides are highly resemble in primary amino acid sequence.Our primary study indicates that the ED50 of Lycosin-Ⅰ and Lycosin-Ⅲ to K562 cancer cell are 7.4ug/ml and 26.2ug/ml respectively,But lycosinⅡ and lycosinⅣ have low inhibition activitiy to K562 cell.Based on the previous study background,we first synthesized and purified these four peptides through immobilized phase chemical synthsis and RP-HPLC method.Maldi-tof MS proved that the mass of synthesized sample is the same of the natural ones and the purity is more than 90%.Then we carried out their cell toxicity scan to K562 cell,and the DNA Ladder tests are completed to further prove the apoptosis of k562 cell, we also tested the change of cytosal free ca2+ concentration to further study if the apoptosis of K562 cell is related to cytoal cacium upregulation.we used flow cytometry to explore the early apoptosis and latter period apoptosis ratio of K562 cell induced by these four peptides and PARP western blot to confirm if the apoptosis is mediated by caspase cascade reaction.The results demonstrated that under high concentration,these four types of cytotoxic peptide can all inhibit K562 cell.but in much lower concentration,only lycosinⅠ and lycosinⅢ have this effect. The ED50 of lycosinⅠand lycosinⅢ to K562 cell is 7.4ug/ml and 26.2ug/ml respectively.Moreover, DNA Ladder test also proved that only lycosinⅠ and lycosinⅢ from Lycosa singoriensis induced K562 cell’s apoptosis in low concentration and lycosinⅡ also induced K562’s DNA fragmentation when its concentration is more than 60ug/ml.On the contrary,lycosinIV can not induce DNA fragmentation although in much higher concentration(more than 120ug/ml).Cellular free cacium test indicated that lycosinⅠ and lycosinⅢ can upregulate the cytosal Free ca2+ concentration.Flow cytometry experiment proved that after incubation with different concentration of lycosinⅠ and lycosinⅢ,many K562 cells are induced to apoptosis program.PARP western blot confirmed that all these four peptides can not induce the shear of PAAP protein,a direct substrate of caspase 3.The result may indicate that the apoptosis induced by lycosinⅠ and lycosinⅢ is inrelavant to caspase cascade reaction,but may mediated by death receptor pathway or endoplastic reticulum apoptosis pathway.