Figure 8. Co-expression of SphK1 and CgA in prostate cancer human samples. Dual immunostaining with anti-SphK1 (brown) and anti-CgA (blue) of a resection specimen harvested in a 84 y.o man with T4NxM1 prostate cancer under complete androgen blockade. Low serum PSA and positive serum NSE suggested poorly differentiated cancer with neuroendocrine features, as confirmed by pathological examination of the resected sample showing high grade prostate cancer (Gleason 9) with positive staining for CgA. Blue granular secretion characteristic of neuroendocrine differentiation is colocalized with brown SphK1 immune reactivity in neuron-like cells (solid arrowhead). doi:10.1371/journal.pone.0008048.g008 SphK1 and Androgen Signaling manner to induce proliferation of epithelial cancer cells and angiogenesis [26]. Although effective, taxane-based chemotherapy for HRPC can delay the fatal outcome by only a few weeks or months, emphasizing the urgent need for new concepts to tackle the hormone-refractory state. By virtue of the uncovered role of SphK1 in mediating the transition from androgen-dependent to androgen refractory state, the pharmacological inhibition of SphK1 and its downstream signaling could represent a viable strategy to prevent or delay the progression to hormone-refractory prostate cancer. Materials and Methods Ethics Statement All animal experiments were conducted as per the guidelines of the European Council Directive 86/609/EEC and approved by the local Inserm Animal Care and Use Committee. Cell Lines and Culture Conditions Human prostate cancer hormone-sensitive LNCaP (DSMZ, Braunschweig, Germany) and C4-2B (Viromed, Minnetonka, MN), and hormone-insensitive PC-3 cells were cultured in RPMI 1640 containing 5% fetal bovine serum (FBS) and passaged less than 15–20 times. FLAG-tagged wild-type human SphK1 cDNA [51] was used for stable transfection of LNCaP cells. Mass pools of stable transfectants were selected in growth medium with 0.8 mg/ ml G418. Empty vector and wild-type SphK1-transfected cells were designated LNCaP/Neo and LNCaP/SphK1, respectively. Stably transfected green fluorescent protein (GFP)-tagged LNCaP and PC-3 cells used for in vivo experiments were maintained in medium containing 0.5 mg/ml G418. For optimal attachment, cells were plated and cultured for 48 h. Thereafter, the medium was replaced by serum-free and phenol red-free medium overnight before the indicated treatments. Materials Culture medium, serum, and antibiotics were from Invitrogen (Cergy-Pontoise, France). Charcoal-stripped serum (CSS) was from Hyclone (Perbio Science, Brebie`res, France). Poly-D-Lysine was from BD Biosciences (Le Pont-De-Claix, France). Dihydro- testosterone (DHT), bicalutamide, forskolin (Fsk), isobutylmethyl- xanthine (IBMX), epinephrine (Epi) were from Sigma. The sesquiterpene quinone F-12509a [52,53] was a gift from Dr Kohama (Daiichi-Sankyo Ltd., Tokyo, Japan). SKI-2 sphingosine kinase inhibitor [29,36] and the PI3Kinase inhibitors, wortmannin and LY294002 were from Calbiochem (Fontenay-sous-Bois, France). [c-32P]-ATP (3,000 mCi/mmol) was purchased from Perkin-Elmer (Courtaboeuf, France), and silica gel 60 high- performance TLC plates were from VWR (Fontenay-sous-Bois, France). Cell Viability Assay Cell viability was carried out using the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) dye reduc- tion assay [54]. Cells were seeded in poly-D-Lysine coated tissue culture plates (6-well) at a density of 36,000 and 14,000 cells/well for LNCaP and PC-3 cells respectively. At the end of the experiments, LNCaP and PC-3 cells were incubated with MTT reagent for 1 and 3 h, respectively. Cell Counting At the indicated times, cells were harvested by trypsinization and resuspended in culture medium. Aliquots were diluted 50-fold in PLoS ONE | www.plosone.org 10 November 2009 | Volume 4 | Issue 11 | e8048