Figure 1.
Intracellular levels of AR, GSK-3β and PSA in different PC cell lines.
(A) Intracellular AR and GSK-3β-protein levels in PC cell lines grown in the absence of androgens. The AR-positive LNCaP, LNCaP-SSR and C4-2 cells as well as the AR-negative PC3 cells were grown for 48 hours under androgen-deprived conditions. Subsequently, cells were lysed and cell lysates were analyzed by Western blotting for AR and GSK-3β. β-actin bands served as loading control. (B) Intracellular PSA levels in CPRC cells grown under androgen-deprived conditions. AR-positive LNCaP, LNCaP-SSR and C4-2 cells were grown and treated as described. Relative PSA levels were determined in the corresponding cell lysates by Western blotting as described in Material and Methods. β-actin bands served as loading control.
Table 1.
Intracellular levels of AR, PSA and GSK-3β in different prostate cancer cell lines.
Figure 2.
SB216763 inhibits phosphorylation of GSK-3β at tyrosine 216.
LNCaP cells and C4-2 cells were seeded into T25 flasks and allowed to adhere overnight. After that, medium was replaced by steroid-free medium and cells were grown for another 24 hours. Subsequently, SB216763 (final concentration 5 µM) was added 30 min prior to DHT (10 nM) treatment. After 4 hours, cell lysates were analyzed for GSK-3βY216 and GSK-3βS9 phosphorylation as described in Material and Methods.
Figure 3.
Inhibition of GSK-3β by SB216763 enhances nuclear export of the AR.
Cells were grown under steroid-free conditions for 24 hours. Cells were then treated with 10 nM DHT and incubated for another 30 min. Subsequently, SB216763 was added (end concentration 5 µM) and cells were allowed to grow for another 210 min. Following this treatment, nuclear extracts were prepared and analyzed for AR and lamin A as described in Material and Methods. AR and lamin A levels were quantified by densitometry. AR levels were expressed in fold-change AR/Lamin of cells grown in absence of DHT and SB216763, which was set at 1.
Figure 4.
Effect of SB216763 on the localization of unliganded AR-Eos fusion protein in LNCaP and C4-2 cells.
LNCaP cells and C4-2 cells were transfected with pAR-t1EosFP and grown for 24 hours in the absence of androgens. Thereafter, cells were treated with/without DHT (final concentration 10 nM). After 4 hours nuclear or cytoplasmic localisation of AR-EosFP was monitored by fluorescence microscopy.
Table 2.
Effects of SB216763 on the nuclear export of the AR.
Figure 5.
Treatment with leptomycin B reverses nuclear export of the AR induced by SB216763 in C4-2 cells.
C4-2 cells grown in the absence of androgens were incubated with/without the CRM1 inhibitor leptomycin B (LMB) 30 min prior to SB216763 treatment. After 4 hours, nuclear extracts were prepared and analyzed for AR and lamin A as described in Material and Methods.
Table 3.
Effect of SB216763 on the nuclear localization and function of the AR in vivo.
Figure 6.
Long term inhibition of GSK-3β in C4-2 cells.
(A) Silencing of GSK-3β in C4-2 cells. C4-2 cells were transiently transfected with pKD-GSK-3β-v1 or pKD-NegCon-v1. 48 hours after transfection, 10 nM DHT was added where indicated. After another 24 hours, nuclear extracts were prepared as described in Material and Methods. (B) Long term inhibition of GSK-3β using SB216763. AR-positive C4-2 cells were treated with increasing amounts of SB216763 for 48 hours in the absence of androgens. Cells were lysed and cell lysates were analyzed by Western blotting for AR. β-actin bands served as loading control.
Figure 7.
Inhibition of GSK-3β by SB216763 inhibits the proliferation in CRPC cells.
AR-positive LNCaP, LNCaP-SSR and C4-2 cells as well as the AR-negative PC3 cells, were treated with increasing amounts of SB216763 for 48 hours (A) or with/without 1 µM SB216763 for 0, 24 and 96 hours (B). Proliferation was measured using an MTT assay as described in Material and Methods. Results shown under (A) are expressed as % of untreated controls ± standard deviation. Results shown under (B) are expressed in percent of SB216763-treated/untreated control which was set at 100% for the time point zero ± standard deviation.
Figure 8.
Inhibition of GSK-3β diminishes nuclear localization and function of the AR in vivo.
The CAM assay was performed with C4-2 cells. Subsequently, nuclear localization of the AR and intracellular PSA distribution in untreated and SB216763-treated tumor-nodules (magnification 400x) was performed as described in Material and Methods. AR-positive cells are marked with stars.