Laschak et al. BMC Cancer 2012, 12:130 http://www.biomedcentral.com/1471-2407/12/130 deaths in the Western world [12]. Current treatment for advanced PCa is mainly based on androgen ablation therapies like orchiectomy, systemic administration of LHRH analog/blocker or anti-androgens. Unfortunately, the benefit of androgen ablation is only transitory. Within a few years many PCa progress to a state of the disease termed castration resistant prostate cancer (CPRC) where tumor cells grow and survive under sub- physiological levels of androgens [13]. Although in vitro the development of a castration resistant phenotype is mostly based on the loss of the AR in PCa cells, several clinical studies demonstrated that the AR is rarely lost in CRPC cells in vivo [14-16]. Indeed, CRPC cells continue to depend on AR-signalling but bypass the requirements for physiological levels of circulating androgens. Various molecular mechanisms that promote AR-dependent growth of CRPC cells growing under androgen deprived conditions have been identified: over- expression/amplification of the AR (hypersensitive path- way), AR mutations that broaden ligand specificity (promiscious pathway), AR-activation by non steroid ligands like growth factors or cytokines (outlaw pathway) [17] as well as the expression of C-terminally truncated AR variants lacking vast parts of the ligand binding domain (LBD). These AR-variants, termed ARΔLBD, are either products of alternative splicing (AR-V), point mutations leading to premature stop codons or proteoly- tic cleavage of the AR protein [18-21]. In contrast to a full length AR which is activated upon androgenic stimuli, previous in vitro studies were able to show that most ARΔLBDs, devoid of a ligand binding domain, are constitutively active [18-21]. As ARΔLBDs lack most parts of the LBD situated in the C-terminus of the AR, they are insensitive to any form of androgen ablation. Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block nuclear receptors via nitrosation of their zinc finger structures in the DNA- binding domain (DBD). The DBD is an essential part of functional full length AR as well as the constitutively active ARΔLBDs. In consequence NO-donors could be of special interest for the treatment of deregulated AR- signalling in CRPC cells. Inhibition of AR-functions following treatment with the long living spontaneous NO- donor (Z)-1-[N-(2-Aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolat (DETA/NO) has recently been demonstrated [22]. However, due to the short half- life of the free radical NO in cell culture medium, rela- tively high concentrations of DETA/NO were necessary to induce effects on intracellular nuclear receptors. In order to generate large amounts of intracellular NO, new compounds, able to deliver NO in a spatially controllable manner, are needed. In our study we used the non-ionic, nitroaromatic diazeniumdiolate JS-K, a glutathione S- transferase(GST)-activated NO-prodrug able to generate Page 2 of 10 high intracellular levels of NO in the micromolar range [23,24]. Studying the effects of JS-K on AR- and WNT-signalling in the AR-positive PCa cell lines 22Rv1 and LNCaP we were able to demonstrate that only a 100-fold lower con- centration of the GST-activated JS-K are necessary to reach comparable biological effects in PCa cells than the commonly used DETA/NO that spontaneously generates NO in physiological fluids. Our data show that NO- prodrugs, capable of generating large intracellular amounts of NO, may be suitable for the treatment of advanced prostate cancer. Methods Plasmids, antibodies, chemicals Probasin promoter driven luciferase-reporter plasmid (pGL3E-Probasin) was kindly provided by Prof. Dr. Z. Culig (Innsbruck, Austria). The pARt1EosFP expression plasmid coding for a green fluorescent AR-Eos fusion protein (AR-EosFP) was a gift from Prof. F. Oswald (Ulm, Germany). The CMV-promoter driven b-catenin expression plasmid pbCAT, containing human b-catenin cDNA with an activating S33Y mutation was provided by Dr. H. Clevers (Utrecht, The Netherlands). TCF- reporter plasmids pTopFlash (TOP) and pFopFlash (FOP), containing three copies of wild-type or mutant TCF-binding sites upstream of a thymidine kinase mini- mal promoter driving a luciferase gene, were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Renilla reniformis luciferase reporter plasmid pRL-tk- LUC was a product of Promega (Mannheim, Germany). Mouse monoclonal antibodies directed against beta actin (ab8224) and nitrotyrosine (ab7048) were products of Abcam (Cambridge, UK). Mouse monoclonal anti- body AR441 directed against the N-terminus of the AR was purchased from Dako (Hamburg, Germany). The NO-donors O2-(2,4-Dinitrophenyl)-1-[(4-ethoxycarbo- nyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K), (Z)-1- [N-(2-Aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO) and the androgen dihydro- testosterone (DHT) were a products of Sigma Aldrich (Taufkirchen, Germany). Cell culture 22Rv1, LNCaP, DU-145 and PC-3 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). LNCaP-SSR, a castration resistant LNCaP subline [25,26], was a generous gift from Prof. Martin Burchardt, Greifswald, Germany. 22Rv1, LNCaP, DU-145 and PC-3 were grown in RPMI-1640 (PAA Laboratories GmbH, Pasching, Austria) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin (Bio- West, Nuaille, France). LNCaP-SSR were routinely grown in RPMI-1640, 1% Penicillin/Streptomycin supplemented