A Preliminary Study On Effect Of Acidic Serine Protease ASP_NJ On Human Chronic Myeloid Cell Line K562

A preliminary study on effect of Acidic Serine Protease ASPNJon human chronicmyeloid cell line K562Objective:Acidic serine protease (ASPNJ) is a protease extracted and purified from Neanthesjaponica, a specific marine life in the northern coast of China. Because of its strongdissolution effect on fibrin and fibrinogen, it was named Neanthes japonica fibrinolyticenzyme, NJF which has an acidic optimal pH. A class of proteases with fibrinolyticactivity such as lumbrokinase showed a remarkable effect on killing many kinds oftumor cells. This effect has attracted the attention of basic and clinical researchers. Asthe effect and mechanism of ASPNJon anti-tumor has not been studied. In this study，we used the chronic myelogenous myeloid cell line K562to mainly observe theinhibitory effect of ASPNJon tumor cells, to clarify the possible mechanism and toexplore new direction and ideas for the clinical application of ASPNJon treatment ofchronic myelogenous myeloid leukemia.Methods:1Chronic myelogenous myeloid leukemia K562cell lines were cultured in vitro asexperimental model;2MTT colorimetric assay were used to measure inhibition of ASPNJon proliferation ofK562cells;3Inverted microscopy, Wright-Giemsa staining and confocal laser scanningmicroscopy, flow cytometry were used to detect the morphological changes andapoptotic effect of ASPNJon K562cells;4For observation of apoptosis related gene expression after exposure of K562cells toASPNJ, Bax mRNA expression was detected by RT-PCR;5To examine the synergistic effect, doxorubicin was combined with ASPNJintreatment of K562cells; 6Hemolysis and aggregation effect of ASPNJon normal human red blood cell werecarried out by routine method and effect of ASPNJon normal human granulocytes wasdetermined by MTT assay.Results:1ASPNJcould inhibite the proliferation of K562cells significanily. The inhibitory rateswere dosage and time dependent;2Morphological observation by inverted microscopy and Wright-Giemsa stainingshowed obvious apoptotic changes including nuclear condensation and nuclearfragmentation, some cells showed significant necrosis, or both apoptotic and necrosischanges;3An typical apoptotic changes such as green cell membrane and red nucleus wereobserved under the laser scanning confocal microscope, while the increased rate ofK562apoptosis were detected by both laser scanning confocal microscopy and flowcytometry;4RT-PCR results showed that Bax gene expression gradually increased withincreasing dose treatment and time exposure of K562to ASPNJ;5There had been a significant tumor suppression synergies when doxorubicinhydrochloride in combination with ASPNJto treat the K562cells;6There were no hemolysis and aggregation effect after exposure of human normal redblood cells to ASPNJ;7There was no significant growth inhibitory effect of ASPNJon normal humangranulocytes.Conclutions：1、ASPNJinhibit the proliferation of K562cells with a certain time and dosagedependent manner. ASPNJcan promote apoptosis and necrosis of K562cells;2、There are significant synergies when doxorubicin hydrochloride in combination withASPNJto treat the K562cells;3、ASPNJmay increase the Bax mRNA expression to induce apoptosis of K562cells ordamage the integrity of the cell membrane to cause K562cells necrosis; 4、ASPNJhas far less effect on normal white blood cells than on K562cells, and it dosenot cause hemolysis and aggregation within six hours.