Fig 1.
Effect of resveratrol in the cell death of ovarian cancer cells OVCAR-3.
A. OVCAR3 cells were treated with 1 to 100 μM resveratrol for 48 h. The cell viability was determined using MTT assays. The data were obtained from an average of four independent experiments. * P<0.05 compared with vehicle control, n = 4/group. B. Representative flow cytometric dot plots for the measurement of apoptosis in OVCAR-3 cells. Resveratrol-induced apoptosis was measured by staining with Sytox Green dye (Y-axis, FL1) and Annexin V-APC (X-axis, FL4). C. Quantitative determination of early apoptotic cell death as the number of annexin V-positive cells with Sytox green-negative cells, and late apoptotic/necrotic cell death as the number of annexin V-positive and Sytox green-positive cells.
Fig 2.
Role of autophagy in resveratrol-induced cytotoxicity in ovarian cancer cells OVCAR-3.
A. Representative western blotting analyses of OVCAR-3 total cell lysates with ATG5, LC3, and p62 antibodies. β-actin and GAPDH were used as loading controls. Three replicate samples were loaded for each treatment group. B. Caspase 3 activation was measured from cell lysates and expressed as the fold change. * P<0.05 compared with the vehicle control, n = 4/group.
Fig 3.
Resveratrol induced apoptosis and autophagy in OVCAR-3 cells at a lower dose.
A. OVCAR-3 cells were treated with 30 μM resveratrol for 48 h, and cell death was determined by flow cytometry. B. Quantitative result of the flow cytometry. C. Representative western blotting analyses of OVCAR-3 total cell lysates with ATG5, LC3, and p62 antibodies with β-actin as a loading control.
Fig 4.
Generation of ROS by resveratrol (30 μM) in ovarian cancer cells OVCAR-3.
A. Intracellular ROS of OVCAR-3 were measured by flow cytometry using H2DCFDA and expressed as the fold change. Resveratrol at 30 μM induced statistically significant ROS. * P<0.05 compared with vehicle control, n = 4/group. B. Oxidative stress marker 4HNE modified proteins were determined using ELISA from the cell lysates of vehicle and Resveratrol-treated (30 μM) OVCAR-3 cells. The quantified modified proteins were expressed as the % of vehicle control samples. * P<0.05 compared with vehicle control, n = 4/group. C. Representative Western blotting showing resveratrol induced elevation of HNE adducts. D. Mitochondrial electron potential change following resveratrol treatment was determined by quantitative flow cytometry and immunofluorescence.
Fig 5.
Resveratrol induced apoptosis and autophagy in Caov-3 cells.
A. Caov-3 cells were treated with 30 μM resveratrol for 72 h, and cell death was determined by flow cytometry. B. Quantitative result of the flow cytometry. C. Representative western blotting analyses of Caov-3 total cell lysates with LC3, and p62 antibodies.
Fig 6.
Pharmacological inhibition of autophagy protects ovarian cancer cells OVCAR-3 against resveratrol-induced cell death.
A. Representative flow cytometry dot diagrams representing each OVCAR-3 cells. Cellular apoptosis was measured in the same method as in Fig 1. Chloroquine reduced the cell killing effect of resveratrol. B. Quantitative determination of total cell death, which was expressed as the percentage of total cells. * P<0.05 compared with vehicle control; # P<0.05 compared with resveratrol-treated samples. n = 4/group. C. Caspase 3 and PARP activities were assayed and plotted as fold change. D. Representative western blotting analyses of OVCAR-3 total cell lysates with LC3, and p62 antibodies.
Fig 7.
RNA interference of ATG5 attenuated resveratrol-induced cell death and caspase activity.
A. ATG5 protein expression was determined from cellular lysates obtained from OVCAR-3 cells loaded with either control random siRNA or ATG5 siRNA. B. Caspase 3 activities were determined from cellular lysates obtained from OVCAR-3 cells loaded with either control random siRNA or ATG5 siRNA. The activities were expressed as the fold change. * P<0.05 compared with vehicle control, # P<0.05 compared with resveratrol-treated samples. n = 4/group. C. Representative western blotting analyses of OVCAR-3 treated with vehicle (Veh) or Resveratrol at 30 μM (RV) with and without siATG5 and total cell lysates were anlyzedwith LC3 and β-actin D. Live cell image from confocal microscope(with 20X objective) using Cyto-Id dye loaded for 15 minutes after treatment with resveratrol at 30 μM in OVCAR-3 cells transformed with random or ATG5 siRNA. E. Quantitative determination of cell death in OVCAR cells using flow cytometry. Resveratrol-induced cell death is attenuated in siATG5-treated and siATG5 plus Z-VAD-FMK (50μM) treated OVCAR-3 cells. * P<0.05 compared with vehicle control; # P<0.05 compared with resveratrol-treated samples. n = 4/group.
Fig 8.
The time course of autophagy and apoptosis induction by 30 μM resveratrol treatment.
A. Live cell imaging by confocal microscopy (with 10X objective) with Cyto-ID dye in OVCAR-3 cells treated with 30 μM resveratrol at 0, 12, 24 and 48 hour time points. B. Live cell imaging by confocal microscopy (with 10X objective) with caspase 3 green detection dye in OVCAR-3 cells at 0, 12, 24 and 48 hour time points. C. OVCAR-3 cells were treated with 30 μM of resveratrol for 12 h, 24 h, and 48 h. Western blotting of total cell lysates were performed with anti-ATG5-ATG12, LC3, and caspase 3 antibodies. No cleavage of ATG5 was observed. The results shown represent an experiment, in which each time-point of treatment was duplicated.