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    include androgen receptor (AR) mutation, AR hypersensi-tivity, and aberrant activation of AR signaling pathways by ligands other than androgens [2]. Accordingly, the cancer remains AR-driven in most patients, a concept that has been proven by the clinical success of abiraterone and enza-lutamide, which have antiandrogenic effects [3−6].
    ADT is implemented today through chemical castration using gonadotropin-releasing hormone (GnRH) agonists or antagonists. Clinical trials have shown that the GnRH ago-nist-induced suppression of luteinizing hormone results in rapid decrease in the levels of testosterone and PSA, whereas a rebound in the follicle-stimulating hormone (FSH) level is observed after initial suppression [7,8]. In contrast, with GnRH antagonists, sustained suppression of both gonadotropins is achieved [7,9]. Despite this potential
    advantage of GnRH antagonists, treatment with agonist is still more frequent.
    The best evidence so far regarding the importance of keeping FSH suppressed has been obtained from clinical and preclinical data showing that increased serum level of FSH correlates with shorter time to CRCaP development, and that FSH receptor (FSHR) expression is found in CaP tissues at a higher level than in benign prostatic hyperplasia and normal prostate tissue [10−15].
    FSHR belongs to the G-protein-coupled receptor gene family [16]. The physiological responses to FSH are medi-ated by different signaling pathways, starting with activation of cyclic adenosine monophosphate (cAMP) and transduced to mitogen-activated protein kinase, extracellular signal-reg-ulated kinase, and the downstream Geranylgeranyl Pyrophosphate cAMP response element-binding protein [17]. Furthermore, FSH has been shown to activate the phosphoinositide 3-kinase and Akt (PI3K/Akt) pathway [18−20]. Activation of this pathway is more likely pertinent to pathological conditions [21].
    Extragonadal expression of the FSHR has been found in the prostate, breast, and blood vessels in a number of malig-nant tumors [12,13,15,22,23], suggesting the involvement of FSHR in the development and progression of multiple cancer types. Our knowledge about the role of FSH in CaP development is yet in its infancy and the mechanisms underlying the conversion of androgen responsive to andro-gen unresponsive state of CaP is poorly understood. In the current study, the effects of FSH on gene and protein regu-lation of human CaP cell lines, representing different stages of the tumor progression from androgen dependence to independence, was investigated by monitoring the expres-sion of the AR target genes PSA and the prostatic tumor suppressor gene NKX3.1 as well as cell proliferation.
    2. Materials and methods
    The human CaP cell lines PC-3, LNCaP, and C4-2 were purchased from the American Type Culture Collection (Manassas, VA) and the immortalized noncancer cells PNT1A from Sigma Aldrich (St. Louis, MO). The authen-ticity of the cell lines was confirmed by Eurofins Genomics (Ebersberg, Germany) before use. The C4-2 cells are derived from LNCaP cells implanted into a male athymic nude mouse. The host was castrated at 8 weeks and a single tumor specimen was excised 4 weeks after castration. Spec-imen was then implanted into a castrated mouse to produce a second generation cell line, C4-2 [24]. This subline was found to be androgen independent and capable of growing in castrated hosts. Cells were routinely grown at 37˚C in a humidified atmosphere of 95% air and 5% CO2 and main-tained in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin-neomycin (Life Technologies, Paisley, UK), and 2 mM L-glutamine (Life Technologies). 
    2.2. Cell treatments
    For protein detection, cells were grown in phenol red-free RPMI medium containing 2% charcoal stripped-serum for 24 hours prior to treatment with FSH (Gonal-F, Merck Serono, Darmstadt, Germany) or 5a-dihydrotestosterone (DHT, Steraloids Inc., Newport, RI) for further 24 hours at concentrations of 50, 100, 200, and 400 IU/l or 10 nM, respectively. For AR induction, PC-3 cells were treated with FSH at concentrations of 400, 800, and 1,600 IU/l. In the mRNA detection, assay cells were treated with 50 and 100 IU/l FSH or 10 nM DHT for 6 or 24 hours. For combination treatment, cells were exposed to 10 mM enzalutamide (Astellas Pharma, London, UK) for 1 hour before the treatment with the aforementioned substances for further 24 hours. Forskolin (Sigma Aldrich, St. Louis, MO) is an agent known to induce cAMP by direct activa-tion of adenylyl cyclase, and was therefore used as an FSH control. In controls, cells were left untreated. For the detection of phosphorylated Akt (p-Akt), cells were pretreated with 10 mM of the PI3K inhibitor, LY294002 (Sigma Aldrich) 1 hour before treatment with FSH at concentrations of 50, 100, and 200 IU/l for 24 hours.