br FSH induced PSA expression in androgen
3.2. FSH-induced PSA expression in androgen independent cells
FSH treatment significantly increased the protein level of PSA and AR in C4-2 7558-80-7 (P < 0.03) at the concentration of 50 IU/l (Fig. 2A). The effect of FSH on PSA induction was less pronounced in LNCaP cells with a nonsignificant trend at concentration of 50 IU/l (Fig. 2B). The significant values were determined by densitometric analysis shown in lower panels. A significant induction of PSA mRNA was also detected in C4-2 cells in response to treatment with 100 IU/l FSH. The induction was observed after 6 hours (P < 0.01) and 24 hours (P < 0.03) of exposure (Fig. 2C). Moreover, in addition to PSA an increase of NKX3.1 gene expression was detected after 24 hours exposure to FSH at concentration of 50 IU/l, but the induction was narrowly short of statistical significance (P > 0.05). No significant induction of PSA mRNA was observed in LNCaP cells treated with FSH (Fig. 2D).
Fig. 1. FSHR expression in CaP cell lines. Upper panels are Western blot analyses of FSHR protein levels in C4-2, LNCaP, and PC-3 cells. Lower panels are qPCR analyses of FSHR transcript level in the aforementioned cells in the absence (ctr) or presence of 100 IU FSH (F100). FSHR = folli-cle-stimulating hormone receptor.
Fig. 2. PSA expression in CaP cell lines. (A and B) protein levels in C4-2 and LNCaP cells. Cells were treated with FSH (F) in the absence or presence of the AR blocker enzalutamide (E) for 24 hours. Lower panels are densitometric analyses of at least 3 Western blot analyses. (C and D) transcript levels of PSA and AR in C4-2 and LNCaP cells treated with FSH or DHT. Expression values were normalized to b-actin and determined by the DDCt method. The y axis shows fold change in arbitrary unit. The values are mean § SD of at least 3 independent experiments. *P < 0.05; **P < 0.01. CaP = prostate cancer; DHT = 5a-dihydrotestosterone; FSH = follicle-stimulating hormone.
Fig. 3. AR and PSA expression in PC-3 cells. (A) Protein levels of PC-3 cells and (B), transcript levels of AR and c-Myc expression in PC-3 cells. Expression values were normalized to b-actin and determined by the DDCt method. The y axis shows fold change in arbitrary unit. The values are mean § SD of at least 3 independent experiments. *P < 0.05; **P < 0.004. F = follicle-stimulating hormone.
3.3. FSH-induced AR expression in PC-3 cells
In PC-3 cells, FSH was able to induce low level of AR protein at a very high concentration of 800 and 1,600 IU/l but not at lower concentrations (Fig. 3A). However, it failed to induce PSA protein in these cells. In contrast, qPCR showed a statistically significantly increased amount of AR (P < 0.004) upon treatment with 800 IU/l of FSH (Fig. 3B). FSH at high concentration also induced c-Myc (P < 0.01) in PC-3 cells.
3.4. Signaling pathway utilized by FSH
FSH enhanced the phosphorylation of Akt in all 3 cell lines to a varying degree, being most pronounced in the PC-3 cells (Fig. 4A). The PI3K/Akt inhibitor, LY294002 abrogated phosphorylation of Akt and reduced the level of PSA induced by FSH in both LNCaP and C4-2 cells (Fig. 4A). Moreover, FSH at concentration of 50 and
100 IU/l also increased the level of b-catenin in PC-3 and C4-2 cells, but not in LNCaP, as compared with con-trols (Fig. 4B). When C4-2 cells treated with the AR blocker, enzalutamide, together with FSH, they
exhibited reduced Akt phosphorylation at concentrations of 50 and 100 IU/l, whereas, at higher concentrations, 200 IU/l, FSH seemed to restore phosphorylation of Akt (Fig. 4C).
Cell proliferation increased when exposed to high FSH concentration in PC-3 cells (Fig. 5A). At concentration of 100 IU/l, PC-3 proliferation was observed after 48 hours, whereas at concentration of 800 IU/l the proliferation started already after 24 hours and continued to the end of the 48 hours experiment (P < 0.03). This was in agreement with the cell viability determined by trypan blue exclusion assay (Fig. 5B). In contrast, DHT treatment did not contrib-ute to PC-3 cell proliferation.
The current study provides experimental evidence that FSH is able to induce PSA expression in CaP cell lines. Moreover, FSH can enhance the PI3K/Akt signaling path-way and induce cell proliferation. The high level of FSHR
Fig. 4. FSH induced Akt phosphorylation and b-catenin expression in CaP cells. Western blot analyses of protein extracts show the phosphorylation of Akt (Ser473) in (A) C4-2, LNCaP, and PC-3 cells after treatment with the FSH (F) in the presence and absence of PI3K/Akt inhibitor, LY294002 (LY); (B) expres-sion of b-catenin in the C4-2, LNCaP, and PC-3 cells; (C) expression of phosphorylated Akt in C4-2 treated with FSH (F) in the absence or presence of enza-lutamide (E). CaP = prostate cancer; FSH = follicle-stimulating hormone.