5.1. Cell Culture

The androgen-dependent PCa LNCaP cell line was provided by the European Collection of Authenticated Cell Cultures (EACC) (Sigma-Aldrich, Saint Louis, MO, USA), whereas the androgen-independent DU-145 cell line was purchased from American Type Culture Collection (ATCC). Cells were cultured under standard conditions in RMPI 1640 and DMEM medium, respectively (Thermo Fisher Scientific Inc/Life technologies, Waltham, MA, USA). Full growth medium was supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS, 1 mM Sodium Pyruvate, 10 mM HEPES buffer and antibiotics (Penicillin 50 U/mL; Streptomycin 50 μg/mL; Neomycin 100 μg/mL). All media and supplements were purchased from Thermo Fisher Scientific Inc/Life technologies, Waltham, MA, USA. An experimental medium was used, i.e., medium serum-deprived without phenol red and antibiotics. Based on our research, the reduction of serum in the medium has no effect on PCa cell viability and proliferation.

ZEA stock solution (0.01M, Sigma-Aldrich, Saint Louis, MO, USA), 2-Phenyl-3-(4-hydroxyphenyl)-5,7-bis (trifluoromethyl)-pyrazolo stock solution [1,5-α]pyrimidine (PHTPP) (1mM, Santa Cruz Biotechnology, Dallas, TX, USA) and (E)-3-(4-t-Butylphenylsulfonyl)-2-propenenitrile stock solution (BAY 117082) (1M, Sigma-Aldrich, Saint Louis, MO, USA) were prepared in methanol and dimethyl sulfoxide (DMSO), respectively. Final ZEA, PHTPP and BAY 117082 concentrations were obtained by dilution in the experimental medium. In all experiments, cells were treated with ZEA (30 µM), ZEA (30 µM) + PHTPP (100 nM), ZEA (30 µM) + BAY (5 µM), ZEA (30 µM) + PHTPP (100 nM) + BAY (5 µM) for 48 h. The concentration of 30 µM of ZEA was chosen on the basis of our previous results [12] and a literature survey. The final concentrations of methanol and DMSO were less than 0.01%; thus, in all experiments as a control (Cnt), cells treated with the experimental medium were used. Cells treated with PHTPP (Cnt PHTPP), BAY (Cnt BAY) and both (Cnt PHTPP BAY) were also used to compare the effects of inhibitors alone.