Biochemical and biological analysis of novel inhibitors of CDC25 phosphatases acting on melanoma cell lines

Cell division cycle 25 (CDC25) enzymes are dual phosphatases that regulate cell cycle progression, being also involved in cell response to DNA damage. Their overexpression has been detected in many tumors, including melanoma. In a previous study compound 19 (NSC 28620) showed a potent in vitro inhibition on CDC25 activity; furthermore, this inhibitor affected the cell viability of some cancer cell lines. In order to identify more potent inhibitors, which possibly functioned as novel anti­cancer drugs, the structure of NSC 28620 was used as a lead compound to synthesize 29 novel molecules. Among them the most active compounds had Ki values in the 0.79 – 10.0 μM interval. Kinetic studies seem to identify two different mechanisms of inhibition, related to specific functional groups possessed by the inhibitors. The cytotoxic effect of the most active compounds on melanoma cell lines was analysed. MTT assay evidenced that at a low concentration, i.e. 10 μM, essentially one compound (cpd 21) significantly reduced the cell growth of melanoma cells in a time­dependent manner. The effect of cpd 21 on cell viability was further confirmed by the colony­forming assay, that showed a similar trend in the ability to inhibit the clonogenicity of melanoma cells. Moreover, the analysis of the levels of various target proteins indicated that cpd 21 provoked an early increase of pCdk1 and a decrease of CDC25C. An arrest of melanoma cells in G2/M of cell cycle, followed by the activation of a caspase­mediated apoptotic program was also observed during the cell treatment with cpd 21. Conversely, cpd 21 didn’t show a cytotoxic effect on BJ­5ta, a nonmalignant fibroblast cell line. Finally, a better investigation on the molecular mechanisms activated by cpd 21 evidenced that the cytotoxic effect of this compound on melanoma cells could be mediated by the reduction of pAkt and an increase of p53 protein levels.