Malignant melanoma is an aggressive disease and its rapidly increasing world-wide. This type of tumor is particularly resistant to conventional chemotherapy and radiotherapy. For this reason in the last years has been developed a variety of a new chemotherapeutic agents. We investigated the action in vitro of four experimental antifolate substances (MR7, MR2, MR36 and NMR707) with a critical target for thymidylate synthase (TS), an essential enzyme for DNA synthesis. These compounds behave as antifolate but show a new structure with respect to all know antifolato agents. We studied how these substances may influence apoptosis in melanoma cell lines SK-MEL-2 (derived from malignant melanoma metastasis) and SK-MEL-28 (derived from primary malignant melanoma).The antifolate agents induced apoptosis in SK-MEL-2 and SK-MEL-28 cells as confirmed by TUNEL technique and Comet Assay. Western-blot analysis showed down regulation of Bcl-2 protein level, up-regulation of p21 and Bax protein level and PARP cleavage especially for MR36, MR7 and MR21. Moreover, these antifolate-induced apoptosis was accompanied by caspase-9 and -8 activation. In addition, a different sensibility for apoptosis was observed for the two cell lines. In fact, in primary melanoma cells, apoptosis occurred also at low concentrations especially for MR7 and MR36. Our results show that TS inhibitors are able to induce apoptosis and for this reason may play a potential role as new therapeutic agents for melanoma, but further studies are necessary to understand their molecular mechanism
