Pentamidine inhibits proliferation in prostate cancer cells. A, Cells were treated with pentamidine (0, 2.5, 5 and 10 μmol/L) for 48 h, and then, cell viability was determined by CCK‐8 assays. Statistical analysis was performed between each prostate cancer cell line and RWPE‐1. B, C, PC3 and DU145 cells were treated with pentamidine (1, 2.5, 5 and 10 μmol/L) for 0, 24, 48 or 72 h, and then, cell viability was determined by CCK‐8 assays. Statistical analysis was performed between cells treated with 1 μmol/L pentamidine and each of the other drug concentration groups. D, E, Long‐term colony formation assays of PC3, DU145 and LAPC4 cells induced by pentamidine (0, 2.5 and 5 μmol/L). (F‐G) Cell cycle distribution of PC3 and DU145 cells incubated with 2.5 μmol/L pentamidine or vehicle for 24 h. Unpaired t test was used for the statistical analysis. *P < .05; **P < .01; ***P < .001; ns, no significance. Data are presented as mean ± SD of at least three independent experiments