Figure 3
Curcumin induces the nonapoptotic and nonautophagic cytoplasmic vacuolation-mediated death of PC-3M cells.
(A): Representative images of phase-contrast imaging and DAPI, AO and MDC staining of PC-3M cells in the presence or absence of 50 μM curcumin for 48 h (upper panel) or under serum starvation for 72 h (lower panel). (B): Transmission electron micrograph of untreated (vehicle) and curcumin-treated (50 μM) PC-3M cells for 24 h at × 2500 (left panel) and × 5000 (right panel), showing cytoplasmic vacuoles devoid of intracellular organelles. (C): Transmission electron micrograph of untreated (vehicle) and curcumin-treated (50 μM) PC-3M cells at higher magnification (× 25,000), showing the dilation of the rough ER. Ribosomes (arrowheads) are attached to some of these membranes, indicating that these vacuoles (*) were derived from the ER (C, upper panel). After 24 h of curcumin treatment, most mitochondria remained unchanged (C, lower panel). (D): The death-inducing effects of curcumin on PC-3M cells were determined by counting the colonies formed. Top: Representative photomicrographs. Bottom: The data are presented as the means ± SD or standard error of 3 independent experiments. ** P < 0.01; *** P < 0.001. (C–F): The PC-3M cells were pretreated with the autophagy inhibitor 3-MA (10 mM) or the caspase inhibitor z-VAD (50 μM) for 1 h and then further treated with 50 μM curcumin for 48 h. (E): Cell extracts were prepared and subjected to western blot for PARP and LC3. (F): Cell vacuolation was observed via crystal violet staining. (G): Cell viability was assessed by performing the MTS assay. (H): Representative photomicrographs of the colony-forming assay.
