JS-K, a glutathione/glutathione S-transferase-activated nitric oxide releasing prodrug inhibits androgen receptor and WNT-signaling in prostate cancer cells

Abstract

Background: 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 consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC).

Methods: Prostate 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.

Results: The 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.

Conclusions: Our 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.

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-K_neg, [C] 2 μM JS-K, [D] 200 μM DETA/NO.

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 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.

Figure 4

NO does not influence hormone-induced nuclear translocation of the AR. Prostate cancer cells (PC-3) expressing a green fluorescent AR-EosFP [37] were incubated for 6 hours in the absence/presence of DHT and JS-K/JS-Kneg. Intracellular localization of AR-EosFP was determined by fluorescence microscopy: (A) untreated controls, (B) 5 nM DHT, (C) 5 nM DHT and 4 μM JS-K, (D) 5 nM DHT and 4 μM JS-K_neg. Bars: Data presented in % cellular localization ± SD. Results are mean values of three independent experiments performed in quadruplicates.

Figure 5

Influence of JS-K and JS-Kneg on the androgen receptor concentration in human prostate cancer cells. 22Rv1 and LNCaP cells were incubated for 30 hours with or without 5 nM dihydrotestosterone in the presence of JS-K or JS-K_neg. Subsequently cells were lysed and separated by SDS-electrophoresis (15 μg protein per lane). Proteins were transferred onto a nitrocellulose membrane. AR and β-actin (loading control) were visualized by immunodetection.

Figure 6

JS-K diminishes WNT-signalling in 22Rv1 cells. 22Rv1 cells were co-transfected with an expression vector for mutated, stabilized β-catenin together with either the TCF reporter construct TOP or FOP as recently described [25]. Data are presented as fold of untreated controls (TOP/FOP) ± SD, **p < 0.01, ***p < 0.001.

Figure 7

Effects of JS-K on the proliferation of prostate cancer cells. (A) 22Rv1 cells are more susceptible to the growth inhibitory effects of JS-K than the androgen sensitive LNCaP cells Cells were cultured in the absence/presence of increasing concentrations of JS-K (1,2,4 μM) or JS-K_neg (4 μM). Cell viability was determined after 96 hours by a colorimetric MTT assay. Results are mean values of four independent experiments performed in quadruplicates. Data are presented as % of untreated controls ± SD, *p < 0.05. (B) Effects of JS-K on the AR negative DU-145 and the AR-positive LNCaP (hormone sensitive) and LNCaP-SSR (castration resistant). Cells were cultured in the absence/presence of increasing concentrations of JS-K (1, 2, 4 μM) or JS-K_neg (4 μM). Cell viability was determined after 96 hours by a colorimetric MTT assay. Results are mean values of four independent experiments performed in quadruplicates.