Analysis of SOD2 activity and its sub-cellular localization during diclofenac-induced apoptosis in melanoma cell lines

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), induces apoptosis in many cell lines through an alteration of the redox state. We have investigated if the pro-apoptotic effect of diclofenac is mediated by an alteration of the redox state, focusing the attention on the involvement of SOD2, an essential mitochondrial antioxidant enzyme. The study was realised using the melanoma cell lines A2058 and SAN, and the human fibroblast immortalized cell line BJ-5ta. Analysis of propidium iodide incorporation and measurement of caspase-3 activity showed an increase of apoptosis after diclofenac treatment in melanoma cells, whereas no effect was observed in BJ-5ta; moreover, the usage of the fluorescent probes DHE and DCFH-DA allowed the demonstration that diclofenac provoked an increase of the intracellular ROS levels only in melanoma cells. Therefore, the effect of diclofenac on the protein levels and enzymatic activity of SOD2 was investigated and only in melanoma cells a significant reduction of these parameters was evident after a 48-h treatment. To investigate if the reduction of SOD2 contributed to the pro-apoptotic effect of diclofenac, the melanoma cells were transfected with siRNAs specific for the SOD2 messenger. In the presence of diclofenac, the SOD2 silencing amplified the proapoptotic effect of the drug, whereas no effect was observed without the drug. To highlight the mechanism by which diclofenac induces apoptosis, we evaluated the mitochondria involvement in this process, through the analysis of the sub-cellular localization of typical mitochondrial proteins. Western blotting analysis showed that in melanoma cells cytochrome c translocated from mitochondria to cytosol after diclofenac treatment. Surprisingly, the drug altered also the sub-cellular distribution of SOD2; indeed, SOD2 appeared in the cytosolic fraction at the expenses of an almost corresponding reduction of this enzyme in the mitochondrial fraction, a feature confirmed by immunofluorescence experiments. In conclusion, the diclofenac exerts its cytotoxic effect selectively in melanoma cells, targeting the mitochondria; in particular, the data suggest that both reduction and cytosolic translocation of SOD2 contribute to the the mithochondrial dysfunction.