Figure 6
Oxidative stress plays a critical role in the curcumin-induced cytoplasmic vacuolation-mediated death of PC-3M cells.
(A): PC-3M cells were treated with 50 μM curcumin for the indicated intervals (0-48 h) and stained with DCF-DA. The total ROS levels were then analyzed via spectrofluorophotometry. Top: Representative photomicrographs. Bottom: Quantification of ROS production as determined by DCF-DA fluorescence. Data are presented as the means ± SD. ** P < 0.01; *** P < 0.001 compared with the control. (B): Fluorescence microscopic images of PC-3M cells treated with 50 μM curcumin in the presence or absence of NAC. Left panel: Representative photomicrographs. Right panel: Quantification of ROS production according to DCF-DA fluorescence. Data are presented as the means ± SD. ** P < 0.01 compared with the control group; ## P < 0.01 compared with the NAC and curcumin cotreatment group. (C): PC-3M cells were pretreated with 10 mM NAC for 1 h and subsequently treated with 50 μM curcumin for 24 h. After cell lysis, the expression of CHOP, LC3 and ubiquitinated proteins was analyzed via western blot. Equal protein loading was confirmed in the western blot by probing for β-actin. (D): Phase-contrast images of PC-3M cells treated with curcumin in the presence or absence of NAC for 48 h and then stained with crystal violet. (E): Cell viability was determined by the MTS assay. Data are presented as the means ± SD of 3 independent experiments. ** P < 0.01; *** P < 0.001. (F): Colony-forming assay. Left panel: Representative photomicrographs. Right panel: Data are presented as the means ± SD of 3 independent experiments. # P < 0.001 compared with the control group; * P < 0.001 compared with the NAC and curcumin cotreatment group. (G-H): Effect of Nrf-2 overexpression on the curcumin-induced cytoplasmic vacuolation (G) and expression of LC3 and ubiquitinated proteins (H).
