Fig. 6.
PUFA influences the interactions of PDK1–AKT and AKT–BAD. (A) C4-2 cells were cultured in medium containing AA or DHA for 48 h. Protein extracts from these cells were immunoprecipitated with mouse anti-PDPK1 or anti-AKT. Western blot analyses were then performed with rabbit anti-AKT, anti-PDK1 and anti-pPDK1S241 antibodies. AKT level was normalized by total PDK1 in the IP-PDK/WB-AKT; PDK1 and pPDK1S241 levels were normalized by total AKT in the IP-AKT/WB-PDK experiment. Normalized levels in DHA-treated samples (white bars) were set at one arbitrary unit. Averages from three repeated experiments and standard deviations are shown. (B) C4-2 and C4-2 BAD cells were cultured in medium containing AA or DHA for 48 h. Immunoprecipitation was performed with mouse antihemagglutinin epitope (C4-2 BAD) or mouse anti-AKT (C4-2), followed by western blotting with rabbit anti-BAD, anti-AKT and anti-phospho-AKT antibodies. AKT level was normalized by total BAD in the IP-BAD/WB-AKT, and BAD level was normalized by total AKT in the IP-AKT/WB-BAD experiment. Normalized levels in DHA-treated samples (white bars) were set at one arbitrary unit. Averages from three repeated experiments and standard deviations are shown (* indicates P < 0.05, Student’s t-test). (C) Knockout of Bad reduced the suppressive effects of ω3 PUFA on prostate cancer. Pten-null mice with or without Bad (Pten − /−;Bad +/+ and Pten − /−;Bad − /−) were fed with ω3 or ω6 diet for 8 weeks. Average mouse prostate weight (mg/25 g body weight, seven mice per group) and error bars are shown. BAD protein expression was confirmed by western blot. (D) Scheme of DHA action. Dietary DHA can be incorporated into sn-2 position of PIP3 to form PIP3 DHA. PIP3 DHA will antagonize PIP3 AA on cell membrane and induce BAD-associated cell apoptosis.