Abstract
BackgroundNitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC).MethodsProstate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay.ResultsThe NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway.ConclusionsOur results suggest that small molecules able to inhibit WNT- and AR-signaling via NO-release represent a promising platform for the development of new compounds for the treatment of CRPC.
Highlights
Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD)
In vitro the development of a castration resistant phenotype is mostly based on the loss of the AR in Prostate cancer (PCa) cells, several clinical studies demonstrated that the AR is rarely lost in castration resistant prostate cancer (CRPC) cells in vivo [14,15,16]
JS-K induces nitrosation of tyrosine residues in cellular proteins In a previous paper NO generated by DETA/NO was shown to nitrosate tyrosine residues in proteins to nitrotyrosine [22]
Summary
Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). Various molecular mechanisms that promote AR-dependent growth of CRPC cells growing under androgen deprived conditions have been identified: overexpression/amplification of the AR (hypersensitive pathway), 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 proteolytic cleavage of the AR protein [18,19,20,21]. 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
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