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

Laschak M, Spindler KD, Schrader AJ, Hessenauer A, Streicher W, Schrader M, Cronauer MV - BMC Cancer (2012)

Bottom Line: Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Urology, Ulm University, Prittwitzstrasse 43, 89075, Ulm, Germany.

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.

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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.
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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.

Mentions: In vitro NO has been shown to repress vitamin D3 signalling 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-homodimerization at concentrations that were already sufficient to inhibit AR-transactivation (Figures 3 and 2).


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

Laschak M, Spindler KD, Schrader AJ, Hessenauer A, Streicher W, Schrader M, Cronauer MV - BMC Cancer (2012)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376035&req=5

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.
Mentions: In vitro NO has been shown to repress vitamin D3 signalling 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-homodimerization at concentrations that were already sufficient to inhibit AR-transactivation (Figures 3 and 2).

Bottom Line: Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Urology, Ulm University, Prittwitzstrasse 43, 89075, Ulm, Germany.

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.

Show MeSH
Related in: MedlinePlus