Figure 9
Involvement of reactive oxygen species in sensitization of TRAIL-resistant LNCaP cells by resveratrol. (A), Generation of ROS by resveratrol. LNCaP cells were seeded in 96-well plates, loaded with 5 μM CM-H2DCFDA dye for 30 min, and treated with either resveratrol (20 μM) or N-acetylcysteine (NAC) (50 mM) plus resveratrol (20 μM) for 0–360 min. Fluorescence was measured by a fluorometer as per manufacturer's instructions (EMD Biosciences/Molecular Probes). * = significantly different from respective controls, P < 0.05. (B), Inhibition of resveratrol-induced caspase-3 activity by NAC. LNCaP cells were pretreated with 50 mM NAC for 2 h followed by treatment with resveratrol (10, 20 or 30 μM) for 12 h, and caspase-3 activity was measured by a fluorometer as per manufacturer's instructions. * = significantly different from respective control; %, # or $ = significantly different from each other, P < 0.05. (C), Inhibition of resveratrol-induced apoptosis by NAC. LNCaP cells were pretreated with 50 mM NAC for 2 h followed by treatment with resveratrol (10, 20 or 30 μM) for 48 h, and apoptosis was measured by TUNEL assay. a, c and e were significantly different from b, d and f, respectively, P < 0.05. (D), Interactive effects of resveratrol and TRAIL on ROS production. LNCaP cells were pretreated with NAC (50 mM) for 2 h followed by treatment with resveratrol (20 μM) with or without TRAIL (50 nM) for 120 min., and ROS production was measured. * = significantly different from respective controls, P < 0.05; # or % = significantly different between groups, P < 0.05. (E), Interactive effects of resveratrol and TRAIL on caspase-3 activity. LNCaP cells were pretreated with 50 mM NAC for 2 h followed by treatment with resveratrol (20 μM) for 12 h, and caspase-3 activity was measured. * = significantly different from respective controls, P < 0.05; # or % = significantly different between groups, P < 0.05. (F), Interactive effects of resveratrol and TRAIL on apoptosis. LNCaP cells were pretreated with 50 mM NAC for 2 h followed by treatment with resveratrol (20 μM) with or without TRAIL (50 nM) for 48 h, and apoptosis was measured by TUNEL assay. * = significantly different from respective controls, P < 0.05; # = significantly different between groups, P < 0.05.