Figure 6

Effects of resveratrol and/or TRAIL on mitochondrial dysfunction. (A), Resveratrol induces the release of cytochrome c and Smac/DIABLO from mitochondria to cytosol. LNCaP cells were treated with or without resveratrol (20 μM) for 12 or 24 h. Cells were fixed, permeabilized and stained with anti-cytochrome c or anti-Smac/DIABLO antibody at 4°C for 18 h. After washing, cells were stained with mitotracker red (mitochondrial staining), DAPI (nuclear staining) and secondary antibody conjugated with FITC (for cyto c or Smac). Red color = mitochondria, green color = cytochrome c or Smac/DIABLO, blue color = nucleus, yellow = co-locolization of cyto c and Smac/DIABLO to mitochondria. (B), Resveratrol releases mitochondrial proteins. LNCaP cells were treated with resveratrol (20 μM) for 0, 6, 12, or 24 h, and cytoplasmic fractions were prepared. Crude proteins were subjected to SDS-PAGE and immunoblotted with anti-cytochrome c (Cyto C), anti-AIF, anti-Smac/DIABLO or anti-Omi antibody. β-Actin was used as a loading control. (C), Interactive effects of resveratrol and TRAIL on the release of mitochondrial proteins. LNCaP cells were treated with resveratrol (20 μM) in the presence or absence of TRAIL (50 nM) for 12 h, and cytoplasmic fractions were prepared. Crude proteins were subjected to SDS-PAGE and immunoblotted with anti-Cyto C, anti-AIF, anti-Smac/DIABLO or anti-Omi antibody. β-Actin was used as a loading control. (D), Effects of Smac siRNA on resveratrol-induced apoptosis. Western blotting data demonstrate that transient transfection of LNCaP cells with Smac siRNA plasmid inhibited Smac protein expression at 48 h. LNCaP cells were transiently transfected with either control plasmid or plasmid expressing Smac siRNA along with plasmid (pCMV-LacZ) encoding the β-galactosidase (β-Gal) enzyme. There was no difference in transfection efficiency between groups. Cells were treated with various doses of resveratrol (0–30 μM) for 48 h, and apoptosis was measured. (E), Enhancement of resveratrol-induced apoptosis by Smac N-7 peptide. LNCaP cells were pretreated with either 25 μM control Smac peptide or Smac N7 peptide for 4 h, and treated with various doses of resveratrol (10, 20 or 30 μM) for 48 h. Apoptosis was measured by TUNEL assay. Data represent mean ± SD. * = significantly different from respective control (P < 0.05).