Laschak et al. BMC Cancer 2012, 12:130 http://www.biomedcentral.com/1471-2407/12/130 soluble tetrazolium bromide into its insoluble formazan derivative by functional mitochondria [32,33]. Statistical Analysis Data are reported as means ± standard deviation. Analy- sis was performed with Student’s T-test (two tailed for independent samples) with p < 0.05 considered as significant. Results 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]. Therefore, we tested JS-K’s ability to nitrosate proteins via intracellular NO-release. In order to generate a control substance without the NO-releas- ing moiety, we took advantage of the observation that sera of patients were repeatedly shown to possess low GST-activity [34,35]. In our experiment 5 μM JS-K was incubated at 37°C in RPMI-1640 supplemented with 10% FBS. Generation of NO in the medium was mea- sured indirectly by the determination of nitrite. Under these conditions maximal medium nitrite levels were detectable after nine to twelve hours (Figure 1A). The remaining inactive JS-K metabolites S-(2, 4-Dinitrophe- nyl)-glutathione and 4-carboxy-piperazine [23] were subsequently used as a negative control and termed JS-Kneg. Incubation with 2 μM JS-K as well as 200 μM DETA/ NO (serving as positive control) led to a nitrosation of intracellular proteins as shown by fluorescescence micro- scopy of nitrotyrosine residues (Figure 1B). In contrast, neither untreated cells nor cells treated with the control solution JS-Kneg harbouring the inactive JS-K metabolites, showed any nitrotyrosine staining (Figure 1B). JS-K inhibits AR-mediated genomic function in 22Rv1 cells To test whether JS-K influences the transcriptional activity of the AR, we transiently co-transfected AR- positive 22Rv1 human prostate cancer cells with pGL3E-Pro, an androgen-responsive probasin-promoter driven reporter plasmid, and pRL-tk-Luc, a Renilla luci- ferase plasmid under control of a constitutively active thymidine kinase promoter (control of transfection effi- ciency). Subsequently cells were incubated for 44 hours with 5 nM DHT and increasing concentrations of JS-K or JS-Kneg. JS-K significantly inhibited DHT-induced AR-transactivation in a dose-dependent manner. Inhibi- tion was already significant at a concentration of 2 μM JS-K. In contrast, JS-Kneg was unable to diminish AR transcriptional activity even at higher concentrations (Figure 2). Page 4 of 10 Figure 1 Release of NO from JS-K. (A) Generation of NO from JS- K in RPMI-1640 medium containing 10% fetal bovine serum was determined indirectly by photorimetric detection of nitrite according to Green et al. [31]. (B) Detection of nitrotyrosine by fluorescence microscopy in 22Rv1 cells: Generation of intracellular NO was detected indirectly by detection of nitrotyrosine (green). Cell nuclei were stained with DAPI. [A] untreated cells, [B] 2 μM JS- Kneg, [C] 2 μM JS-K, [D] 200 μM DETA/NO. JS-K does not modulate dimerization or nuclear localization of the AR In vitro NO has been shown to repress vitamin D3 sig- nalling through inhibition of vitamin D receptor (VDR) - retinoic X receptor (RXR)-heterodimerization [36]. To test whether JS-K can inhibit AR-homodimer formation in vivo, we performed an AR-specific mammalian two hybrid (M2H)-assay in AR-negative PC-3 and DU-145 cells (Figure 3, Additional File 1). As seen in Figure 3, JS-K was unable to repress DHT-induced AR-homodi- merization at concentrations that were already sufficient to inhibit AR-transactivation (Figures 3 and 2). In order to test whether high NO concentrations are able to influence the nuclear translocation of the AR, we transfected the AR-negative PC-3 with a construct coding for a green fluorescent AR-Eos fusion protein (AR-EosFP) [22,37]. Subsequently cells were grown for six hours in the presence/absence of 5 nM DHT with or without JS-K or JS-Kneg. Under hormone-free conditions the AR was pre- dominantly located in the cytoplasm (> 80%) whereas in presence of DHT the AR was transported to the nucleus