ABSTRACT
Objective To investigate the effect of docosahexaenoic acid (DHA) combined with cyclooxygenase-2 (COX-2) selective inhibitor NS-398 on the apoptosis of cholangiocarcinoma QBC939 cells and the mechanism.Methods In vitro,cholangiocarcinoma QBC939 cells were treated with 0,15,30,45,60 and 75 μg/ml DHA with 0,25,50,100,150 and 200 μmol/L NS-398,respectively.The absorbances of the QBC939 cells were measured by CCK8 and its growth inhibition ratios were analyzed.Flow cytometry was applied to detect cell apoptosis.The level of β-catenin and COX-2 mRNA and protein were measured by real-time PCR,immunocytochemistry and enzyme-linked immunoadsordent assay,respectively.Results DHA combined with NS-398 could significantly suppress the growth of QBC939 cells (P ＜ 0.05).When the concentration of DHA went up to 45 μg/ml and NS-398 was 100 μmol/L,the relative growth inhibition rate of QBC939 cells was 90.0％.If the concentrations were increased,the result showed no significant differences.Furthermore,flow cytometry analysis indicated that DHA combined with NS-398 could induce QBC939 cells apoptosis at the early stage,and the apoptosis rate was significantly different between the experimental and control groups (P ＜ 0.01).Real-time PCR showed low β-catenin and COX-2 expression in QBC939 cells disposed by DHA combined with NS-398,and their expression were significantly different between the experimental and control groups (P ＜ 0.01).Immunocytochemistry and ELISA demonstrated that DHA combined with NS-398 could decrease β-catenin and COX-2 protein expression in QBC939 cells.Conclusion DHA combined with NS-398 induced apoptosis and inhibited proliferation of cholangiocarcinoma cells QBC939 in vitro through targeting β-catenin and COX-2.