Figure 5

XN induces ROS production in PCa cells. The antioxidant NAC abrogates all the biological effects of XN. Time course of DU145 and PC3 cells treated with 10 µmol/L XN and then stained with H₂DCFDA to assess intracellular ROS levels show a maximal ROS induction after 30 min that is maintained up to 24 h (A). Pretreatment for 90 min with NAC (10 mmol/L) completely inhibited ROS induction by XN (10 µmol/L) at 30 min (B). DU145 and PC3 cell growth inhibition in the presence of 5 and 10 µmol/L XN for 48 h is completely abolished by cotreatment with 10 mmol/L NAC (C). Means ± SD are shown (*P < 0.05, **P < 0.01, ***P < 0.001). PC3 cell migration in the presence of 10 µmol/L XN, evaluated in a 24-h wound healing assay, is restored by 10 mmol/L NAC (D). Western blot analyses of FAK and AKT phosphorylation in DU145 and PC3 cells exposed for 5 h to 10 µmol/L XN in the presence of NAC (alone or in combination) indicates that ROS inhibition restores the basal phosphorylation level of both kinases, even in the presence of XN (E). The ELISA test (PC3 cells are shown) and Western blotting analysis in both cell lines showed that pretreatment with NAC abolished the inhibitory activity of 10 µmol/L XN (5 h treatment) on NF-κB in TNF-α (10 ng/mL) stimulated cells; NAC alone produced marginal effects (F). ELISA data are shown as means ± SD (***P < 0.001). Western blot representative results from one of two independent experiments are shown. C (within panels A, B, and D), control; OD, optical density.