Cytotoxic effect of diclofenac in the melanoma cell lines A2058 and SAN

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID) widely used in clinical therapeutics, has cytotoxic effects and induces apoptosis in many cultured cell lines. Many studies suggest that NSAIDs, and in particular the highly selective cyclooxigenase-2 inhibitors, could act as anticancer. We have investigated the involvement of mitochondrial dysfunction in the mechanism of diclofenac-induced apoptosis in the melanoma cell lines, A2058 and SAN, and in the human fibroblasts immortalized cell line BJ5ta. The analysis by phase contrast microscopy of diclofenac-treated cells showed the typical morphologic changes related to apoptosis only in A2058 and SAN cell lines. These effects appeared after a treatment with 150 lM diclofenac suggesting a preferential cytotoxic effect in the two melanoma cell lines. Propidium iodide incorporation followed by cytometric analysis showed an increase of apoptosis during diclofenac treatment. Since the caspase 3 represents the final effector of the caspase dependent apoptotic process we have also analysed its activity following diclofenac treatment. A marked increase of caspase 3 activity in the cell extracts of A2058 and SAN cells was evidenced by spectrofluorimetric analysis as well as the analysis of intracellular levels of bcl-2 confirmed the pro-apoptotic effect of diclofenac only in the melanoma cell lines. Furthermore, the cytotoxic effect of diclofenac in the melanoma cell lines was associated to a decrease of the antioxidant SOD2 protein levels. These preliminary data suggest that mitochondria could represent a diclofenac target and in particular the reduction of SOD2 protein contributes to the mitochondria dysfunction.