Effection Of Cathepsin D Expression Levels On Human Lymphoma Cell Line Raji Proliferation And Chemosensitivity To Adriamycin

ObjectiveCathepsin D(Cath-D) is a lysosomal acid proteinase.In the range of pH 2.8～5.0,it can degrade structural and functional proteins,peptides,peptide precursors and hormone. Furthermore,it plays an important active role in the renewal,remodelling a wide variety of tissue and in apoptosis.In recent years,much study has showed that the change of Cath-D expression levels in a variety of tumor cells(breast cancer,ovarian cancer,human glioma, laryngeal cancer,lung cancer,colorectal cancer,leukemia,lymphoma,etc.) is closely related to proliferation and apoptosis.Our study aims to explore the expression of Cath-D in human lymphoma cell lines Raji and it's correlation with Raji cells proliferation and Chemosensitivity to Adriamycin(ADM).Methods1.Cell lines and cell culture Human lymphoma cell line Raji which was purchased from the Shanghai Cell Bank,was cultured in suspension in RPMI-1640 medium containing 10%fetal bovine serum at 37℃in a humidified atmosphere of 5%CO₂.The cells were divided every 2～3 days to maintain exponential growth.2.Methods Raji cells in logarithmic phase will be divided into 5 groups:control group,namely,Raji cells without any treatment;â‘ dimethyl sulfoxide(DMSO) control group,an equivalent DMSO control group established because of experimental Pepstatin A (PepA) dissolving in organic solvent DMSO;â‘¢PepA treated group,Raji cells 100μmol/L PepA alone for 16h;â‘£ADM treated group,Raji cells treated with 3.15μg/mL(48h 50% inhibiting concentration(IC50)of ADM) alone for 48h;⑤PepA+ADM treated group, namely,Raji cells pre-treated with 100μmol/L PepA for 16h,and then treated with 3.15μg/mL ADM for 48h.Methyl thiazolyl tetrazolium(MTT) assay was used to analyze proliferation curve of different densities of Raji cell in order to find the appropriate cell density for detection 48h proliferation activity of Raji cell.The Raji cell proliferation inhibition rate of different groups was analyzed by MTT assay,the 48h ADM's IC50 concentrations used as working concentration in this experiment.Changes of Cath-D expression level in each group before and after ADM treatment analyzed by Western blot assay.After pre-treated with PepA,Raji cells to ADM sensitivity analyzed by MTT assay.3.Statistics All data were analyzed by DPS V3.11 Professional Edition statistical software and represented as(?)±s and percentages.Analysis of variance was used for much inter-group comparison,T-test was used for comparison between the two groups, Spearman Level test was used for two correlation parameters.Results1.In the 72h period,cells growth and time were showing a linear relationship when cell density in the range of 1×10³ï½ž5×10⁴/mL(Figure 1).When the cell suspension density was 5×10³/mL,OD value of 72h determined by MTT assay was in the range of confidence interval,between which and time was a good linear relationship,therefore,this density was appropriate for detecting 48h proliferative activity of Raji cell.2.ADM's working concentration Raji cells after treated by ADM with concentration of 1,2,4,8,16μg/mL respectively for 48h,the inhibition rates were(17.59±4.26), (44.20±6.05),(64.61±2.52),(73.82±3.32) and(79.36±1.21)%.48h ADM's IC50 was 3.15μg/mL,and this drug concentration was used for working concentration.3.Change of Cath-D expression level before and after treatment by reagents of each group Cath-D was expressed in Raji cells,compared with blank control group,Cath-D expression level of DMSO control group had no significant changes,Cath-D expression decreased in both PepA treated group and PepA+ADM treated group,the level of the decline of the two groups no statistical difference(P＜0.05);Cath-D expression of Raji cells in ADM treated group had increased(P＜0.05).4.Effort of different Cath-D expression level on Raji cell proliferation MTT showed that,no change in Raji cells proliferation in both DMSO control group and PepA treated group,while Raji cell proliferation were significantly inhibited in ADM treated group and PepA+ADM treated group,cell proliferation inhibition rate of PepA +ADM treated group was significantly lower than that of ADM treated group(P＜0.05).Conclusions1.Compared with blank control group,there was no change in Cath-D expression levels of Raji cells in DMSO control group(P＜0.05),it showed that this experiment used doses of DMSO had no significant effect on the results of the experiment.Cath-D expression levels in both PepA treated group and PepA+ADM treated group were decreased,indicating that this experiment used in doses of PepA can effectively inhibit the Cath-D expression of Raji cells.2.Compared with the blank control group,the Cath-D expression of PepA treated group decreased,but Raji cell proliferation was no change in the group(P＜0.05), indicating in the absence of chemotherapy drug ADM,changes of Cath-D expression level in Raji cells no effect on proliferation and apoptosis.3.Compared with blank control group,Cath-D expression of PepA+ADM treated group decreased,that of ADM treated group increased;Raji cell proliferation of both ADM treated group and PepA+ADM treated group was significantly inhibited,while the cell proliferation inhibition rate of PepA+ADM treated group was significantly lower than that of ADM treated group(P＜0.05),which indicated that Cath-D may be involved in ADM-mediated apoptosis of Raji cells and can enhance the chemosensitivity of Raji cells to ADM.Cath-D may be an ideal target for Cancer gene therapy.