Laschak et al. BMC Cancer 2012, 12:130 http://www.biomedcentral.com/1471-2407/12/130 Page 7 of 10 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-Kneg (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-Kneg (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. off-target effects). Interestingly, the castration resistant LNCaP-SSR cells were more susceptible to the growth inhibitory effects (growth inhibition at 1 μM JS-K 31 ± 2%, p < 0.01) than the parental LNCaP cells (growth inhi- bition at 4 μM JS-K 33 ± 1%, p < 0.01) (Figure 7B). In contrast to the AR-positive LNCaP and LNCaP-SSR the AR-negative DU-145 were less susceptible to the growth inhibitory effects of JS-K (growth inhibition at 4 μM JS-K 17 ± 9%). Discussion Current treatment for advanced PCa is mainly based on androgen ablation therapies like chemical or surgical cas- tration or application of anti-androgens. Although hor- monal therapy is initially very effective, the benefit of androgen withdrawal is only transitory. While still expressing high levels of functional AR, some PCa cells acquire the ability to grow and survive under castrate levels of circulating androgens. The mechanisms involved in an altered AR-signalling of CRPC cells include an over-expression/amplification of the AR (hypersensitive pathway), point mutations broadening ligand-specificity of the receptor (promiscuous pathway) as well as activa- tion of the AR by peptide growth factors or cytokines (outlaw pathway) [17]. A recently identified mechanism enabling CRPC cells to bypass the requirements for phy- siological levels of androgens is the expression of C-term- inal truncated, constitutively active AR splicing variants termed AR-V. Due to the loss of the C-terminus, AR-Vs are lacking essential parts of the ligand binding domain [21]. In the absence of androgens, AR-Vs were shown to induce AR-signalling either as AR-V/AR-V-homodimers or AR-V/AR-heterodimers [18,48]. Whereas AR-V/AR-V homodimers are unaffected by hormone ablation in gen- eral, some AR-V/AR-heterodimers were insensitive to the commonly used anti-androgens [48]. Given the fact that both, AR wild type as well as AR-V, play a crucial role in a high percentage of CRPC there is an urgent need for an AR-blockade that does not involve the LBD. Nitric oxide, a free radical gas, has been repeatedly shown to impair the function of nuclear receptors via tar- geting their cys4-type zinc fingers, located in the DBD of these receptors [49]. This is well exemplified by studies showing a NO-induced nitrosation of the cystein residues within the zinc finger structures of steroid receptors like the estrogen receptor (ER) [50]. As the DBD is a crucial part of functional full length AR as well as constitutively active AR-Vs, it is tempting to speculate that NO-donors able to release NO in a spatial and timely controllable manner could be of special interest for the treatment of deregulated AR-signalling in CRPC cells. Inhibitory effects of NO on AR-function in PCa cells was first described in vitro using the spontaneous NO- donor Deta/NO [22]. In a recent clinical study Siemens et al. were able to show an increase of the PSA doubling time in patients treated with the NO-donor glyceryl trini- trate (GTN) [51]. No cardiovascular toxicities or serious side effects were encountered during treatment with GTN [51]. Although the number of patients enrolled in the study was relatively low (29 patients), our data and the clinical data with GTN [22,51] support the assump- tion that NO might be suitable for the treatment of advanced PCa. Commonly used NO-Donors like Deta/NO, sponta- neously dissociate in a pH dependent process thereby releasing the free radical NO. Unfortunately NO exhibits only a relatively short half-life in the tissue culture med- ium or body fluids. Therefore, we tested a potent NO- prodrug, the non-ionic nitroaromatic diazeniumdiolate