Anti-proliferative effects of AKR1C3 inhibitors in various prostate cancer cell lines. (A) 22Rv1 cells were treated with increasing concentrations of the natural chalcones MF-11, MF-14, and MF-15 dissolved in RPMI + 10% CS-FCS over 5 days or (B) with 10 µM MF-15 over various time points. (C) DuCaP, DuCaP EnzaR and 22Rv1 cells were seeded into 96-well plates and treated with MF-15 (10 µM), indomethacin (indo, 20 µM), AKRi (50 µM), enzalutamide (enza, 5 µM) or a combination of enzalutamide (5 µM) and MF-15 (10 µM) over 5 days as described under material and methods. Cell viability was measured through colorimetric MTS cell viability assay (Promega) and indicated as percentage of mock control (DMSO). Data represent the mean ±SEM from three independent experiments. Statistical comparisons to the mock control were expressed with an asterisk (* p < 0.05, ** p < 0.01, *** p < 0.001), comparisons to enzalutamide with a hash key (#). (D) Western blot analysis of AKR1C3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) of DuCaP, DuCaP EnzaR, and 22Rv1 cells. Representative images of blots were taken with Image Studio software (Li-Cor).