Figure 4

MTA upregulated p53 expression, via DNA damage and intracellular ROS accumulation (A) p53 expression was assessed by RT-PCR after 24 h treatment with 0.84 μM MTA. Raw data was extracted by the ΔΔCt method and expression results are represented as fold change relative to the expression of not exposed cells (control). MTA induced an increment of p53 expression comparing with controls. This increment was reduced when intracellular ROS levels were blocked with 10 mM NAC. These results are the mean of three independent experiments. (B) The role of p53 in cell cycle progression was verified by HT144 cell cycle distribution analysis by flow cytometry after inhibition of p53 transcriptional activity by PFT. Results are represented as the percentage of cells in S-phase. MTA-untreated cells (control) are represented in black, PFT-only treated cells in green, 0.17 μM MTA-only treated cells in blue and cells treated with both PFT and 0.17 μM MTA in red. MTA-induced cell cycle arrest at S-phase was avoided when inhibiting p53 activity. (C) DNA damage was analysed by Comet assay. Cells were exposed to 0.84 μM MTA for 24 or 48 h, after which double/single strand breaks were apparent. Less damage was observed when intracellular ROS levels were inhibited with 10 mM NAC. Illustrated fields are representative; magnification, 40 x. (D) MTA increased the level of ROS production. Intracellular ROS levels were measured by flow cytometry. Cells were exposed to 0.84 μM MTA for the indicated times before incubation with DCFH-DA for 60 min at 37°C. A significant increase in ROS production was apparent after 18 h in all melanoma cell lines. The graph illustrates the behaviour of the A375 cell line. (E) Cells were pretreated with 10 mM NAC before 0.84 μM MTA exposure. MTA-induced cytotoxicity was found to be inhibited in all employed melanoma cell lines when ROS was blocked. Results are expressed as the mean of three independent experiments, each one performed in triplicate.