Figure 5. DHA prevents PGE₂-induced dissociation of Axin/GSK-β complex in HCC cells.

(A) PGE₂ induces dissociation of Axin from GSK-3β in Hep3B cells. The cells were treated with different concentration of PGE₂ in the serum-free medium for 1 hr. The cell lysates were obtained for immunoprecipitation with Axin antibody followed by immunoblotting with antibodies against GSK-3β and Axin. (B) The time dependent effect of PGE₂ on Axin/GSK-3β dissociation. Hep3B cells were treated with PGE₂ (10 µM) for different time points in the serum-free medium. The cell lysates were obtained for immunoprecipitation with anti-Axin antibody followed by immunoblotting with antibodies against GSK-3β and Axin. (C) DHA prevents PGE₂-induced Axin/GSK-3β dissociation. Hep3B cells were treated with PGE₂ (10 µM) or vehicle in the absence or presence of DHA (60 µM) in serum-free medium for 1 hr. The cell lysates were obtained for immunoprecipitation with anti-Axin antibody followed by immunoblotting with antibodies against GSK-3β and Axin. (D) DHA inhibits PGE₂-induced TCF/LEF reporter activity. Hep3B cells were transiently transfected with TCF/LEF-Luc reporter vector. After transfection the cells were treated with PGE₂ with or without DHA in the serum-free medium for 24 hr. The cell lysates were obtained to determine the luciferase activity. The data are presented as mean ± SD of six independent experiments. DHA treatment significantly decreased TCF/LEF reporter activity (*p<0.01 compared to control; **p<0.01 compared to PGE₂ treatment).