Effects of platelet-activating factor and its differential regulation by androgens and steroid hormones in prostate cancers

Abstract

Background: Platelet-activating factor (PAF) is an arachidonic acid metabolite that plays an important role in cell proliferation, migration and neoangiogenesis, but whether it is involved in the progression of prostate cancer remains undiscovered.

Methods: Clinical prostate specimens were investigated with immunohistochemistry method and in vitro cell experiments referred to MTS cell proliferation assay, invasion and migration experiment, quantitative real-time RT-PCR assay, western blotting analysis and ELISA assay.

Results: Platelet-activating factor synthetase, lyso-PAF acetyl transferase (LPCAT1), increased significantly in castration-resistant prostate cancer (CRPC) specimens and CRPC PC-3 cells than that in controls. Intriguingly, PAF induced invasion and migration of PC-3 cells but not LNCaP cells. The PAF receptor antagonist inhibited proliferation of LNCaP and PC-3 cells. Dihydrotestosterone (DHT) treatment caused a decrease in LPCAT1 expression and PAF release in LNCaP cells, which could be blocked by androgen receptor antagonists. Finally, DHT increased LPCAT1 expression and PAF release in PC-3 cells in a Wnt/β-catenin-dependent manner.

Conclusion: For the first time, our data supported that PAF might play pivotal roles in the progression of prostate cancer, which might throw a new light on the treatment of prostate cancer and the prevention of the emergence of CRPC.

Figure 1

Immunolocalisation of LPCAT1 in human prostate cancer specimens. (A) Representative H&E staining of a clinical prostate cancer sample (Gleason score 4+3=7). (B) Protein expression of LPCAT1 in the same prostate cancer sample (Gleason score 4+3=7) by immunohistochemistry: LPCAT1 protein expression of prostate adenocarcinoma cells was strong positive (long arrow), whereas that of normal prostate gland epithelial cells was negative or weak positive (short arrow). In a paired prostate cancer sample, LPCAT1 protein expression of tumour cells significantly increased from (C) ADT-naive biopsy specimen (Gleason score 3+4=7) to (D) their matched CRPC specimen (Gleason score 4+4=8). Original magnifications × 400 (A–D).

Figure 2

Effects of PAF and a specific PAF receptor antagonist ABT-491 on the proliferation of prostate cancer cells. (A) Expression of PAF receptor (PAF-R) in LNCaP cells and PC-3 cells was determined by western blot. (B) Prostate cancer cells were exposed to increasing concentrations of ABT-491. (C) Prostate cancer cells were treated with increasing concentrations of PAF for 24 h. Values are presented as mean percent control±s.e.m. of six different experiments. *P<0.05 and **P<0.01 compared with control.

Figure 3

Effects of PAF and ABT-491 on invasion and migration of PC-3 cells. (A, C) Effects of increasing concentrations of PAF on invasion and migration of PC-3 cells. (B, D) Effects of increasing concentrations of ABT-491 on invasion and migration, and pretreatment with ABT-491(10⁻⁵ M) on PAF-induced invasion/migration of PC-3 cells. Data are presented as mean percent control±s.e.m. for five cultures (n=5) performed in triplicate. *P<0.05 and **P<0.01 compared with control; ^#P<0.05 and ^##P<0.01 compared with PAF treatment alone (10⁻⁷ M).

Figure 4

Effects of DHT on LPCAT1 mRNA and protein expression and PAF release in prostate cancer cells. Cells were treated with increasing concentration of DHT, and in combination of DHT (10⁻⁷ M) and RU486 (10⁻⁶ M) or flutamide (10⁻⁶ M) for 24 h. The mRNA and protein levels of LPCAT1 in LNCaP cells (A) and PC-3 cells (B) were measured by real-time RT–PCR and western blot analysis, respectively. Representative protein bands were presented on the top of corresponding histogram. Concentrations of PAF in LNCaP (C) and PC-3 (D) cell culture were determined by ELISA. Time course expression of LPCAT1 in LNCaP cells (E) and PC-3 cells (F) treated by DHT-CMO-BSA are also shown. Flu, flutamide; Ru, RU486.Values are presented as mean percent control±s.e.m. (A, B, E, F) or mean±s.e.m. (C, D) for five cultures performed in triplicate. *P<0.05 and **P<0.01 compared with control; ^#P<0.05 and ^##P<0.01 compared with DHT (10⁻⁷ mol/l⁻¹).

Figure 5

Effects of 17β-oestradiol on LPCAT1 mRNA and protein expression and PAF release in prostate cancer cells. Cells were treated with 17β-oestradiol (10⁻⁷ M) in the presence or absence of RU486 (10⁻⁶ M), MPP (10⁻⁶ M) and cyclofenil (10⁻⁶ M) for 24 h. (A, C) LNCaP cells and (B, D) PC-3 cells. Values are presented as mean percent control±s.e.m. (A, B) or mean±s.e.m. (C, D) for five cultures performed in triplicate. *P<0.05 and **P<0.01 compared with control; ^#P<0.05 and ^##P<0.01 compared with 17β-oestradiol (10⁻⁷ mol/l⁻¹).

Figure 6

Dihydrotestosterone-induced PAF release in β-catenin knockdown PC-3 cells. (A) Representative bands for protein expression of β-catenin in cells transfected with β-catenin siRNA. (1, 2) Cells transfected with Neg-siRNA; (3, 4) cells transfected with β-catenin-specific siRNA. (B) Effects of β-catenin knockdown on PAF release in PC-3 cells in the presence or absence of DHT (10⁻⁷ M). Values are presented as mean±s.e.m.; n=4. *P<0.05 and **P<0.01 compared with negative control siRNA; ^##P<0.01 compared with DHT (10⁻⁷ M) treatment in cells transfected with negative control siRNA. (C) Representative bands for protein expression of β-catenin in LNCaP cells and PC-3 cells. Cells were treated with DHT (10⁻⁷ M) in the absence or presence of flutamide (10⁻⁶ M) for 24 h (LNCaP and PC-3 cells) or 48 h(PC-3 cells). C (control), 1 : 10 000 ethanol in PBS; D, DHT; F, flutamide.