Figure 3

Restored AR expression in LNCaP^RANKLcells grown in 3-D suspension increases cell adhesion to ColI. (A) IHC analysis indicates that the suppressed AR expression in LNCaP^RANKL cells could be restored, when the cells were grown as a xenograft tumor or in 3-D suspension culture. Arrows denote AR nuclear localization. (B) Restored AR in LNCaP^RANKL cells grown in 3-D suspension was confirmed with western blotting. While AR expression is high in LNCaP^Neo cell grown in 2-D monolayer or 3-D suspension, LNCaP^RANKL cells express AR protein only under 3-D suspension conditions. (C) The restored AR is biologically functional since it could promote PSA promoter activity as detected by a PSA promoter-luciferase reporter assay, which showed 4.3-fold increased activity in 3-D suspension culture compared to the LNCaP^RANKL cells in 2-D monolayer. Each black bar represents relative light unit (RLU) as a fold difference of 3-D suspension/2-D monolayer for each cell line. (D) The function of the restored AR protein in regulating cell adhesion was indicated by treating LNCaP^RANKL cells with the synthetic androgen R1881 (10 nM, 48 hours), which resulted in significantly enhanced cell adhesion to ColI. On the other hand, the antagonistic effect of the anti-androgen casodex (20 nM) was seen mainly under 3-D suspension culture conditions. Each value is the mean ± SD of 2 independent experiments done in triplicate.