Laschak et al. BMC Cancer 2012, 12:130 http://www.biomedcentral.com/1471-2407/12/130 Page 5 of 10 Figure 2 JS-K inhibits AR-dependent reportergene activity in 22Rv1 cells. Cells were cotransfected with a probasin reporter gene plasmid and a Renilla luciferase plasmid serving as transfection control. Subsequently cells were grown in presence/absence of 5 nM DHT. Reportergene activity was determined using the Dual Luciferase Assay System from Promega. Data are presented as fold of untreated controls ± standard deviation (SD). Results are mean values of three independent experiments performed in quadruplicates: *p < 0.05; **p < 0.01. (Figure 4). As seen in Figure 4 addition of JS-K (final con- centration 4 μM) was unable to reverse the nuclear locali- zation of the AR in DHT treated PC-3 cells (82% ± 2% to 78 ± 2%). In a complementary experiment transfection of AR negative DU-145 cells with AR-EosFP yielded similar results (Additional File 2). JS-K leads to a down-regulation of the AR-protein In contrast to the commonly used spontaneous NO-donor DETA/NO which generates NO in aquaeous solutions, JS-K is able to generate high amounts of NO within target cells after enzymatic activation by GST. The profound dif- ferences in local NO-production prompted us to analyze the effects of JS-K on the AR-concentration in the prostate cancer line 22Rv1 and LNCaP. Both cell lines express functional AR-proteins. Whereas 22Rv1 cells express a 120 kDa AR-isoform as well as a C-terminally truncated, constitutively active 79 kDa AR-V splicing variant [38] the LNCaP cells express a full length AR (119 kDa) with a Figure 3 JS-K does not inhibit AR-dimerization in presence of androgens. AR-dimerization was determined in PC-3 cells using the CheckMate Mammalian Two-Hybrid System from Promega as described in Material and Methods. Results are mean values of four independent experiments performed in quadruplicates. point mutation at position 877 (T877A) [39]. Treatment with JS-K for 30 hours diminished the amount of all AR- forms in 22Rv1 as well as in LNCaP cells (Figure 5). The downregulation of the AR in LNCaP was less pronounced than the dramatic decrease of AR and AR-V in 22Rv1 cells (Figure 5). In both cell lines incubation with JS-Kneg had no influence on intracellular AR-levels (Figure 5). JS-K inhibits WNT/b-catenin signaling in 22Rv1 cells Interaction of the canonical WNT/b-Catenin pathway with the AR is thought to promote progression of PCa to the terminal castrate-resistant stage [40]. Crosstalk between the canonical WNT and AR-pathways occurs at several levels: (a) b-Catenin interacts with the AR to increase its transcriptional activity [27], (b) WNT-signal- ling influences AR-signalling through its ability to regu- late AR-mRNA as well as AR-stability [41]. Several lines of evidence indicate that NO is involved in the regulation of the WNT/b-catenin pathway in various tumors [42-44]. A common approach to analyze the cano- nical WNT-pathway in vitro is the over-expression of mutated stabilized b-catenin (S33Y) that activate TCF/ LEF-dependent reporter gene constructs [27]. As LNCaP cells are unable to activate b-catenin dependent reporter gene expression [27] we tested the effects of JS-K on the canonical WNT-pathway in 22Rv1 cells. As seen in Figure 6, JS-K was able to diminish b-catenin signalling in the CRPC cell line 22Rv1 in a dose dependent manner. Down- regulation of the canonical WNT-pathway was already significant at JS-K concentrations of 1 μM (reduction of transcriptional activity: 42 ± 8%, p < 0.01). Growth inhibitory effects of JS-K are most pronounced in castration resistant prostate cancer cells Based on our observations we tested the antiproliferative effects of JS-K on prostate cancer cell lines. JS-Kneg served as negative control. Cellular proliferation was assessed by