Figure 3.

ROS signalling involvement in melatonin cytotoxic effect. (A) Ewing's sarcoma cells were treated with melatonin 1 mM at the indicated times. Intracellular peroxides were measured by flow cytometry and expressed as % fluorescence vs vehicle-treated group (0 h); ^*P<0.05 vs vehicle-treated cells; ^#P<0.05 vs group with highest value. (B) SK-N-MC cells were incubated with or without melatonin 1 mM, Trolox (TRX) 100 μM, ascorbic acid (ASC) 200 μM or the combination for 72 h. Cell viability was determined by the MTT reduction assay and cell number after the combined treatments was expressed as % vs each single treatment group; ^*P<0.05 vs each single treatment group, ^#P<0.05 vs melatonin 1 mM group. (C) Melatonin activates NF-κB pathway. NF-κB DNA-binding ability analysed by EMSA. SK-N-MC cells were incubated with 1 mM melatonin at the indicated times: (P), labelled probe; (C), control for specificity (100-fold excess of unlabelled probe). Histogram represents densitometric analysis from at least three independent experiments; ^*P<0.05 vs vehicle-treated group (0 h). (D) The inhibitor of NF-κB activation parthenolide prevents cell death induced by melatonin 1 mM. Cells were treated for 72 h with melatonin with or without 5 μM parthenolide, and cell death was evaluated as the LDH release. ^*P<0.05 vs vehicle-treated cells, ^#P<0.05 vs melatonin 1 mM group. (E) The inhibitor of NF-κB activation parthenolide prevents upregulation of Fas and Fas L induced by melatonin 1 mM. Cells were treated with melatonin, with 5 μM parthenolide or with their combination, and Fas and Fas L expression was evaluated by quantitative PCR. GAPDH was used as a housekeeping gene. Relative gene expressions are represented as the n-fold increase compared with basal level (dotted lines represent vehicle-treated cells). ^*P⩽0.05 vs vehicle-treated cells. ^#P<0.05 vs melatonin 1 mM group.