Figure 1.

Fas/Fas L expression after treatment with melatonin. (A) SK-N-MC cells were treated with or without 1 mM melatonin for 8 or 24 h, and TNFR1, TNF-α, Fas, Fas-L, DR4, DR5 and TRAIL expression were determined by quantitative PCR to assess mRNA expression levels. GAPDH was used as a housekeeping gene. Relative gene expressions are represented as the n-fold increase compared with basal level (vehicle-treated cells: dotted line). ^*P<0.05 vs vehicle-treated cells. (B) Expression of Fas and Fas L proteins was determined by western blot after treatment with 1 mM melatonin for the indicated times. GAPDH was used as housekeeping gene. A representative blot is shown and densitometric analysis of the immunoblots of three independent experiments is represented below. ^*P<0.05 vs vehicle-treated group (0 h). (C) Flow cytometric analysis (FASCS) of SK-N-MC cells treated with 1 mM melatonin for 48 h. Cells were stained with anti-FAS (left panel) or anti-FAS-L (right panel) and then PE-conjugated secondary antibody. The values obtained are shown above the graphs. ^*P<0.05 vs control (vehicle-treated group). (D) Overexpression of FAS/FAS-L mediates melatonin cytotoxic effect. SK-N-MC cells were incubated with 1 μg ml⁻¹ of neutralising antibody against FAS (ZB4), FAS-L (NOK1) or both, 4 h prior to the addition of melatonin 1 mM. Cell death was evaluated by the release of lactate dehydrogenase (LDH) after 72 h. ^*P<0.05 vs vehicle-treated cells; ^#P<0.05 vs melatonin 1 mM-treated group. (E) Evaluation of cell death by the release of LDH after 72 h of incubation with melatonin plus 0.1 μM of nordihydroguaiaretic acid (NDGA); ^*P<0.05 vs vehicle-treated group; ^#P<0.05 vs melatonin 1 mM-treated group.