Table 1.
Primers and Probes for qRT-PCR.
Table 2.
RSV and FIDAS down-regulate activity of AR-dependent reporter-gene activity in LNCaP and 22Rv1 cells.
Table 3.
Effects of RSV and FIDAS on AR- and AR-V7-mRNA-expression in prostate cancer cell lines.
Figure 1.
Regulation of AR- and ARΔLBD-protein by RSV and FIDAS in human PC cell lines.
Prostate cancer cells were grown in presence/absence of DHT and incubated with RSV or FIDAS for 24 hours. Subsequently, intracellular AR or ARΔLBD-levels (AR-V7, Q640X) were determined by Western Blotting as described in Material and Methods: (A) LNCaP, (B) 22Rv1, (C) PC-3 transfected with full length AR, (D) PC-3 transfected with Q640X.
Figure 2.
Effects of FIDAS and RSV on AR and Q640X signalling.
PC-3 cells were transiently co-transfected with AR or Q640X, the pARE(2x)-luc reporter and the pRL-TK control plasmid as described in Material and Methods. (A) PC-3 cells transfected with AR were grown with/without 5 nM DHT in the presence/absence of RSV or FIDAS for 24 hours. Data are expressed as % transactivation of DHT stimulated AR ( = % activity ARDHT/ARbasal) which was set at 100%; *p<0.05. (B) PC-3 cells transfected with constitutively active Q640X were treated with RSV or FIDAS for 24 hours. Data are expressed as % transactivation of Q640X which was set at 100%, *p<0.05).
Figure 3.
Effects of RSV and FIDAS on AR/Q640X-signalling under androgen deprived conditions.
PC-3 cells were transiently transfected with AR, Q640X or AR+Q640X, together with pPSA-61luc and pRL-TK reporter plasmids as described in Material and Methods. Cells expressing Q640X and/or AR were grown for 24 hours in presence/absence of RSV (100 µM) or FIDAS (50 µM). Data are expressed in fold AR basal activity which was set to 1. As depicted, RSV and FIDAS significantly decreased PSA-promoter mediated reporter gene activity in Q640X and AR co-expressing cells, *p>0.05.
Figure 4.
RSV and FIDAS do not inhibit nuclear translocation of AR and Q640X.
PC-3 cells were transiently transfected with expression plasmids coding for green fluorescent AR-EosFP or Q640X-EGFP fusion proteins as described in Material and Methods. Subsequently cells were treated with/without 5 nM DHT in presence/absence of 100 µM RSV for 120 minutes. Nuclear localization of fluorescent AR or Q640X proteins was analyzed by fluorescence microscopy.
Table 4.
Effects of RSV and FIDAS on the nuclear translocation of AR and Q640X.
Figure 5.
Effects of RSV and FIDAS on the dimerization of AR and Q640X.
Formation of AR and Q640X homo/hetero-dimers was analyzed 24 hours after RSV or FIDAS treatment using a M2H as described in Material and Methods. Within this time frame RSV as well as FIDAS did not exhibit a significant in vitro toxicicity (see Table S1). AR/AR dimers: Formation of AR/AR homodimers was analyzed in PC-3 cells grown under androgenic stimuli (5 nM DHT) in presence/absence of RSV or FIDAS (FIDAS/RSV untreated + DHT = 100%). AR/Q640X and Q640X/Q640X dimers: Formation of AR/Q640X heterodimers (AR-VP16/ACT and Q640X-GAL4/BIND) or Q640X/Q640X homodimers was analyzed in the absence of DHT in RSV/FIDAS treated/untreated PC-3 cells (FIDAS/RSV untreated was set at 100%), *p<0.05.
Figure 6.
Effects FIDAS on prostate cancer micro-tumors growing on the CAM.
CAM assays were performed with PC-3 (AR negative) and LNCaP (AR-positive) as described in Material and Methods. Proliferation of PC cells was determined by nuclear staining of KI67. AR-activity was analyzed by PSA-staining.
Table 5.
Effects of RSV and FIDAS on on PCa cells in vivo.