Laschak et al. BMC Cancer 2012, 12:130 http://www.biomedcentral.com/1471-2407/12/130 JS-K. The compound was shown to be activated by glu- tathione S-transferase (GST), a key phase II detoxifica- tion enzyme that is frequently over-expressed in cancer tissue. Among the various isoforms of GSTs which are located in the cytoplasm and the mitochondria, mainly the π isoform is able to form the so called < Meisenhei- mer complex >[52] which finally delivers NO directly within the cell. First experimental in vivo studies using tumor xenografts in a mouse model, showed that JS-K exhibits a strong anti-tumor activity and is generally well tolerated by the host organism [5,23,24]. In order to test the effects of JS-K on AR signalling, we treated the AR-positive CRPC cell line 22Rv1 grown in presence of DHT with increasing concentrations of JS-K. Generation of intracellular NO was monitored indirectly by detection of nitrotyrosine using fluorescence micro- scopy. JS-K was able to inhibit the genomic functions of the AR in 22Rv1 cells. The inhibition of AR-signalling was neither due to an inhibition of AR-dimerization nor to an inhibition of nuclear translocation. Interestingly, JS-K was able to diminish intracellular levels of all AR-isoforms in PCa cells. However, in contrast to the dramatic downregu- lation of AR-V and AR in 22Rv1 cells the intracellular AR levels of LNCaP were only slightly affected. So far, studies on intracellular steroid receptor levels following NO-treatment are sparse. In a previous study using Deta/NO as an NO-donor the concentration of the endogenous AR in LNCaP remained unaffected [22]. In contrast, DETA/NO was able to downregulate the intra- cellular levels of the Drosophila melanogaster ecdysteroid receptor (EcR) when overexpressed in chinese hamster ovary cells, CHO-K1 [53]. Although the heterologeous expression of an insect steroid receptor in CHO-K1 cells differs largely from the situation found in LNCaP cells that express high levels of intracellular AR under physio- logical conditions the present data suggest that there are cell specific differences in the sensitivity of cells towards NO-treatment. Moreover, the generation of NO by JS-K largely depends on the intracellular GST-activity that dif- fers between different cell lines or cell types. The fact that NO is able to inhibit the canonical WNT- pathway in different tumor cells [42-44] including pros- tate cancer cells (as seen in this study) offers a more intriguing explanation for the massive down regulation of AR and AR-V in 22Rv1 cells. There is experimental evi- dence that the WNT/b-catenin-pathway is able to up- regulate AR-mRNA expression in prostate cancer cell lines through interaction with TCF/LEF binding sites situated in the promoter region of the AR [41]. In con- trast to the highly aggressive castration resistant 22Rv1 cells which are able to drive a WNT-typical T-cell factor (TCF)-dependent reporter gene activity, the androgen sensitive LNCaP are unable to do so [27]. In conse- quence, the inhibition of the canonical WNT-pathway Page 8 of 10 would lead to a decrease of AR and AR-V mRNA in 22Rv1 cells but not or to a lesser extent in LNCaP. In proliferation assays castration resistant 22Rv1 and LNCaP-SSR cells were more susceptible to the growth inhibitory effects of JS-K than LNCaP. Whereas LNCaP cells largely depend on androgenic stimuli, the 22Rv1 and LNCaP-SSR cells are able to grow under androgen deprived conditions. LNCaP cells express a full length AR with a point mutation at position 877 (T877A), enabling the AR to be stimulated by different steroids (promiscuous AR) [39]. In contrast to the hormone dependent LNCaP, the castration resistant LNCaP-SSR cells were shown to exhibit high levels of nuclear AR in the absence of hormonal stimuli [26]. Increased nuclear AR-levels were paralleled by elevated PSA-levels sug- gesting that the AR is active in these cells [26]. In 22Rv1 cells two AR-forms can be found: A larger AR- form expressing 3 zinc finger motifs due to the duplica- tion of exon 3 (AREx3dup) and a C-terminally truncated, constitutively active AR-V [20]. In the absence of andro- genic stimuli, some AR-Vs have been shown to form constitutively active AR-V-homodimers or AR/AR-V- heterodimers, thereby uncoupling the need of CRPC cells for physiological levels of androgens [18,48]. Although it is unknown whether AREx3dup is able to form androgen independent heterodimers with AR-V in 22Rv1 cells, its expression of 3 zinc finger structures makes it probably also more susceptible to the effects of NO. The fact that NO targets the zinc finger structures of wild type AR, mutated AR-forms as well as AR-Vs suggests that NO-donors are promising compounds for the treatment of deregulated AR-activity. Moreover, the observation that CRPC cells like 22Rv1 or LNCaP-SSR, expressing either constitutively active AR-Vs or func- tionally deregulated full length AR, are more susceptible to the effects of JS-K than LNCaP cells further supports this assumption. Conclusions From our in vitro studies we conclude that GST-acti- vated NO-prodrugs like JS-K show features which might be suited for the treatment of advanced PCa. Therapies targeting the AR and the canonical WNT pathway may lead to a more efficient treatment of CRPC. Therefore, compounds like JS-K may serve as leader compounds for the development of NO-based therapeuticals, target- ing key structures in castration resistant prostate cancer cells. Additional material Additional file 1: Figure 1S: JS-K does not inhibit AR-dimerization. Complementary data to experiment presented in Figure 3: AR- dimerization was determined in DU-145 cells using the CheckMate