Inhibition And Possible Mechanisms Of Acidic Serine Protease On Lymphocytic Leukemia Cell Jurkat

Objectives:Acid Serine Protease (Acidic Serine Protease from Neanthes japonica, ASPNJ) isa kind of fibrin strong dissolution Protease which was isolated and characterized inDepartment of Biochemistry from Coastal animal Neanthes japonica, was named theNereid fibrinolytic enzyme, but now named acid serine protease (ASPNJ). We havedone a lot of experiments on leukemia cells in earlier period, and found that ASPNJcan induce cell apoptosis, inhibit the proliferation of leukemia cells. In thisexperiment we has worked on the effects of ASPNJon various cells and exploredthe mechanism of ASPNJespecially on Jurkat cell. The study of the anti-tumor effectsof ASPNJ in vitro on Jurkat cell can provide a new method for research and treatment,and may provide an innovation direction for research and treatment of leukemia.Methods:1MTT was used to measure inhibition of ASPNJon these cells(K562，RK562,NB4,HL-60,239T THP-1,U937, Jurkat);2Wright-Giemsa staining (16μg/ml) was used to detect the morphologicalchanges of Jurkat cells;3Jurkat cells were treated with ASPNJand cell membrane protein wereextracted, and2D image were obtained through IEF and SDS-PAGE;4Image-master2D Platinum7.0software was used to analyze gel images, and thedifferential protein spots were identified between control cells and the ASPNJtreatedcells;5Significant different protein spots were selected to undergo mass spectrometryanalysis to identify the specific proteins;6Western Blot and RT-PCR were used to confirm the2D and MS results. Result:1According to the date from MTT,we have found that the inhibitory rates ofASPNJon a variety of leukemia cells were dose dependent and the inhibitory rateon Jurkat cells was obvious;2Wright-Giemsa staining showed the cells morphological changes, such as thechromatin condensation and nuclear pyknosis, and obvious apoptosis;3The2D images of Jurkat cell membrane protein spots were highly matchedbetween three gels from both the control group and the experiment group; the specificinformation of different protein spots were obtained through Mass spectrometryanalysis database exploring. The results revealed that.14-3-3theta，Ranbp-1and Btf3were reduced in the experiment group. The results of Western Blot showed theconsistency of the change of the expression of three kinds of protein showed in2Dimages, while the results of RT-PCR showed that the differences of the three kinds ofprotein in mRNA level is not obvious.Conclusion:ASPNJinhibits the proliferation of Jurkat cells significantly with dosedependent manner. ASPNJcan induce Jurkat cell apoptosis and necrosis. Thedecreased expression of14-3-3theta protein, Ranbp-1protein and Btf3protein causedby ASPNJmay relate with the mechanism of inhibitory effects of ASPNJon Jurkat cells.