Figure 5. DCPIP induces oxidative stress in human melanoma cell lines.

(A) Induction of intracellular oxidative stress in human A375 melanoma cells by treatment with DCPIP. Cells were exposed to DCPIP (10, 20, 40 µM, 24 h) and intracellular oxidative stress was assessed by 2’,7’-dichloro-dihydrofluorescein diacetate staining followed by flow cytometric analysis. One representative experiment of three similar repeats is shown. (B) Antioxidant protection against DCPIP-induced caspase-3 activation. Cells were pretreated with NAC (10 mM, 24 h) or left untreated. After medium change, DCPIP (40 µM) was added and caspase-3 activation was examined after another 24 h by flow cytometric detection using an Alexa Fluor 488-conjugated monoclonal antibody against cleaved procaspase-3. One representative experiment of three similar repeats is shown. (C) Modulation of intracellular glutathione content in A375 and G361 melanoma cells exposed to DCPIP (40 µM, 6h) in the absence or presence of DC (60 µM). Total glutathione content was normalized to protein content. (D) For NQO1 knockdown, G361 melanoma cells were treated with siNQO1- or siControl or left untreated as described in Materials and Methods. Intracellular glutathione content was then determined in G361 melanoma cells exposed to DCPIP (40 µM, 6h). Total glutathione content was normalized to protein content (mean ± SD, n=3).