Fig. 3.

Effect of bioactive compounds on ROS production and on antioxidant protein levels in prostate cancer cells. LNCaP and PC-3 cells were incubated with melatonin (250, 500, 1000 µM), silibinin (25, 50, 100 µM), curcumin (10, 25, 50 µM) or resveratrol (25, 50, 100 µM) for 30 min and stained with MitoSOX red (A, B) or DHE (C, D) for 30 min. Representative histogram of MitoSOX fluorescence shift after curcumin incubation in shown in the top corner of figures A and B. ROS levels were measured by flow cytometry and are presented as the percentage fold change relative to control cells. LNCaP (E) and PC-3 (F) were cultured with 1 mM melatonin, 50 µM silibinin, 25 µM curcumin or 50 µM resveratrol for 48 h and CuZnSOD and catalase protein levels were assessed by western blot. β-Actin was employed as loading control. Experiment was repeated at least 3 times and a representative experiment is shown. CuZnSOD/catalase protein ratio was calculated in LNCaP (G) and PC-3 (H). (I) Effect of bioactive compounds on H₂O₂ release in prostate cancer cells. Cells were cultured in complete medium at a density of 75×10⁵ cells/ml and incubated with 1 mM melatonin, 50 µM silibinin, 25 µM curcumin or 50 µM resveratrol for 24 h. An electro-oxidation method was used for H₂O₂ determination. (J) PC-3 cells were exposed to resveratrol (25, 50, 100 µM) and ROS production was measured using DCFH2-DA after 3,6 or 24 h incubation. Data are shown as mean ±S.E.M of three independent samples. *p<0.05; **p<0.01; ***p<0.001 versus CON. Experiment was repeated 3 times and a representative experiment is shown.