Figure 5. Bicalutamide inhibits cell proliferation and SphK1 stimulation triggered by DHT. Overnight serum deprived LNCaP cells were incubated under 5% CSS conditions and treated with or without 10 mM bicalutamide in presence or not of 10 nM DHT for 2 (A, top) or 6 days (A, bottom). DNA synthesis and cell counting measurements were expressed as percent of control at the beginning of treatment. B, overnight serum deprived LNCaP cells were incubated with 10 nM DHT for 30 min in presence or not of 10 mM bicalutamide, and SphK1 activity was quantified and expressed as percent of control at the beginning of treatment. Columns, mean of at least five independent experiments; bars, SD. The P values between the means are as follow: ***, P,0.001; **, P,0.01; or ns, non significant. doi:10.1371/journal.pone.0008048.g005 SphK1 and Androgen Signaling DHT with regard to cell proliferation (Fig. 9A, left panel) or secretion of PSA (Fig. 9A, right panel). However, the NE-like phenotype is reversible upon replenishing the media with complete serum during several weeks [20]. When LNCaP androgen- depleted for 42 days were switched back to normal FBS medium, cell growth resumed over the period of the following week and, within the next 21 days in FBS medium, the cell morphology reverted to that of parental LNCaP cells (data not shown). As demonstrated in Fig. 8B, these cells were now not only able to grow normally and secrete PSA in presence of FBS like parental LNCaP (Fig. 1A), but also to respond again to the addition of DHT under CSS conditions similar to parental cells (Fig. 4C and D). Interestingly, the reversability induced by replenishing the media with complete serum for 21 days was associated with a profound down-regulation of SphK1 activity (Fig. 9C), which went back to the levels found in parental cells. These data suggest that SphK1 could represent an adaptable mechanism by which NE like-cells might survive in an environment deprived from androgens. Discussion Compelling evidence has implicated SphK1 in promoting oncogenesis and response to anticancer therapies. On the one hand, SphK1 expression is up-regulated at the message or protein level in various solid tumors [29,30,31,32,33], and has been linked with poor survival for brain and breast cancers [30,34]. On the other hand, anticancer treatments (chemotherapies, radiation therapy) trigger down-regulation of SphK1 activity in various cancer cell and animal models suggesting that SphK1 could act as a ‘‘sensor’’ to anticancer therapies particularly in prostate cancer [36,37]. Here, we explored the potential role of SphK1 in the androgen- controlled cell growth and survival of the hormone-sensitive LNCaP prostate cancer cell model. As formerly established, androgen deprivation reduced cell growth and PSA secretion of LNCaP cells. Simultaneously, SphK1 activity was significantly down-regulated. The observed SphK1 inhibition was not relying on de novo synthesis of ceramide (data not shown), which has been previously reported to accumulate following androgen ablation in LNCaP cells [48]. Moreover, neither oxidative stress nor cathepsins - which have been previously implicated in SphK1 activity inhibition caused by other stimuli in various cell systems [49,50] - seemed to be involved (data not shown). The short-term efficacy of androgen depletion was confirmed in vivo using an orthotopic model of xenotransplanted LNCaP cells where castration could not only noticeably reduce tumor size and metastasis dissemination but also induce a significant SphK1 inhibition. Of note, in hormono-insensitive PC-3 cells, neither androgen deprivation in vitro nor castration in vivo had any effect on tumor growth and SphK1 activity. SphK1 overexpression markedly inhibited the antiproliferative effect resulting from androgen removal. These results emphasize other findings establishing that enforced expression of SphK1 displays a cytoprotective effect against various stimuli such as chemothera- peutics notably in prostate cancer cells [36]. On the other hand, restoring the androgen environment was associated with a surge in SphK1 activity. The addition of dihydrotestosterone (DHT) induced a rapid and ephemeral activation of SphK1 suggestive of a non-genomic effect. The PI3K/Akt cascade has also been reported to participate in the rapid effects of DHT through a direct interaction between a membrane associated androgen receptor and PI3K [39,40]. Herein, we demonstrate that SphK1 stimulation occurs down- PLoS ONE | www.plosone.org 7 November 2009 | Volume 4 | Issue 11 | e8048