Study On Mechanism Of Anticancer Activities Of Acidic Serine Protease (ASP_NJ) In Leukemia Cell Lines By Proteomics Approach

Objectives:An Acidic Serine Protease, ASPNJis isolated and characterized in our lab fromNeanthes japonica in north coastal area of China. It was previously named fibrinolyticenzyme because of its strong effect on fibrin dissolution. We have recently reportedthe anti-tumor activities of ASPNJin leukemia cell lines. ASPNJcan induce apoptosis,inhibit the proliferation of acute myeloid leukemia NB4cells and chronic myeloidleukemia K562cells, and promote the sensitivity of chemotherapeutic drugs. Toexplore the mechanism by which ASPNJplay anticancer activities in leukemia cells,membrane proteomics study on K562cells and NB4cells treated with ASPNJwascarried out. This study may provide a new way to investigate the pathogenesis andtreatment of leukemia.Methods:K562cells and NB4cells were treated with ASPNJ(16μg/mL) for24h, andcell membrane proteins were extracted, quantitated and used to performtwo-dimensional electrophoresis; Gel images were analyzed by usingImage-master2D Platinum7.0software, the differential protein expression pattern wasobtained from the control group and the experimental group, and protein spots weredetermined by mass spectrometry and specific information of protein was identifiedfrom database. RanBP1and14-3-3theta proteins were selected for furtherconfirmation by Western Blot and RT-PCR detection.Result:A large number of membrane proteins of both K562and NB4cell were welldistributed, and the gel background was clear. Image-master analysis of2D Platinum7software was used to display the differential protein pattern between the control andexperimental cell groups, the matching rate was80%, and protein spots with Ratio≥2.0were considered as significance. Specific information obtained through mass spectrometry analysis on differentially expressed proteins include many proteinsinvolved in cell proliferation, division, molecular chaperones, signal transductionmolecules, ATP synthesis and transport-related proteins. Two proteins RanBP1and14-3-3theta were significantly reduced in drug-treated groups of both cell lines.Expression trend of RanBP1and14-3-3theta protein between treated and untreatedcell groups were further confirmed by Western Blot analysis; while RT-PCR analysisshowed that the mRNA levels of these two proteins were not changed withsignificance between treated and untreated cell groups with ASPNJ. RanBP1functionsin cell nuclear transport and spindle formation.14-3-3theta protein may be associatedwith cell signal transduction pathways by interaction with other proteins and may playother functions. Recent studies have shown that these two proteins are respectivelyinvolved in anti apoptosis effect on cells. Research reports on RanBP1and14-3-3theta proteins in relation with leukemia were rare.Conclusion:Our data suggest that the mechanism of effects of ASPNJon myeloid leukemiacells such as apoptosis induction and inhibition of cell proliferation, as well aspromotion of cell chemo-sensitivity may involved in reduction of some differentiallyexpressed membrane proteins or membrane associated proteins including RanBP1and14-3-3theta, which may become the therapeutic target protein of myeloid leukemia.ASPNJmay provide a new therapeutic approach for leukemia.