Mechanism Of Lycosin-I Selectively Inhibiting The Chronic Myelogenous Leukemia K562 Cell Line

Spider venom,serving as a weapon of both predation and defense,is a considerable source of natural pharmacological active Substances.Spider venoms comprise a vast array of bioactive peptides that have specific pharmacological properties and targets.Compared with proteins,bioactive peptides from spider venom become most promising pharmacological tools and drug leading molecules,by virtue of its small molecular weight contributing to the easiness of structural analysis,stability to resist degradation in vivo as well as its strong plasticity.In this work,we first purified and then chemically synthesized a cationic peptide(named as Lycosin-?)with antitumor activity from the venom of Lycosa singoriensis,which displays linear facultative a-helical conformation other than ICK motif that is commonly found in most spider neurotoxins.In order to estimate its antitumor effect,cancer cells of diverse types were screened.Hematologic malignancies cells,solid tumor cells,clinical leukemia patients' cell,as well as cell lines from normal person were treated with Lycosin-I respectively,followed by CCK8 and MTT experiments to determine its ability to inhibit cell proliferation and cytotoxicity.K562 cells were set as an antitumor model,upon which differential proteins of before and after-the treatment of Lycosin-I was found based on comparative proteomics approach of 2-dimensional electrophoresis combined with mass-spectrometric technique.bioinformatic analysis suggested that some proteins are related to AKT signaling pathway,then the changes of associated genes and proteins were verified by qPCR and western blots.Cells were co-incubated with Lycosin-I and AKT pathway activator IGF-1 or AKT pathway inhibitor LY294002 respectively to assess its inhibition capability to cell proliferation.The results suggested high selectivity of Lycosin-I against different types of tumor cells,which was proved by the fact that it has the IC50 of 2.011?M against K562 cell after 24h treatment yet almost no effect on normal cell lines even if they were treated with the concentration of 50?M.And the selectivity also exists in its interaction with dissimilar types of clinical leukemia patient cells,among which strong inhibition,with the IC50 of less than 10?M,was observed in the case of chronic myelogenous leukemia cells.AKT signaling pathway-related proteins such as FGR,PHB,PCNA,VDAC2,and TCL1A were identified through comparative proteomics motheds in addition to ones concerning cellular metabolism,degradation,stress response.AKT pathway inhibitor LY294002 could enhance the activity of Lycosin-I upon K562 while AKT pathway activator IGF-1 could redeem the inhibitory effect on K562 cell proliferation.qPCR indicated the down-regulation of PI3K and up-regulation of PTEN on the upstream of AKT signaling pathway,and down-regulation of c-MYC and up-regulation of VDAC2 on the downstream of AKT signaling pathway.Western blot revealed the downregulation of p-AKT while total AKT amount remained stable.These experiments demonstrated the inhibition of K562 cells proliferation is relevant to AKT signaling pathway.