TSI INDUCES APOPTOSIS WITH A P53 INDIPENDENT PATHWAY IN MELANOMA CELL LINES AND IN NORMAL HUMAN MELANOCITES

Melanoma patients have a very poor prognosis and this type of tumor is particular resistant toconventional chemotherapy and radiotherapy. For this reason in the last years has beendeveloped a variety of new therapeutic agents. We investigated four experimental antifolatesubstances (MR7, MR36, MR21 and NMR707) with a critical target for thymidylate synthase(TS), an essential enzyme for DNA synthesis and repair. All the compounds were tested fortheir inhibitory properties against TS enzymes from different species: Lactobacillus casei,Pneumocystis carinii, Cryptococcus neoformans and human. These compounds behave asantifolates but show new structure with respect to all known antifolate agents. We studiedhow these substances may influence apoptosis in melanoma cell lines SKmel 2 (derived frommalignant melanoma metastasis) and SKmel 28 (derived from primary malignant melanoma)and normal human melanocytes. The antifolate agents induced apoptosis in Skmel 2 andSkmel 28 cells. These results were confirmed by Comet Assay. Western blot analysis showeddown regulation of the level of Bcl2 and PARP clivage especially for MR36, MR7 and MR21.Moreover a different apoptotic behaviour for the two cell lines was observed, in fact inprimary melanoma cells apoptosis occurred also at low concentrations especially for MR7.After chemotherapeutic treatment with all the tested compounds p53 was not modulatedindicating a p53 independent pathway for apoptosis. Caspases 8 and 9 clearly mediateantifolate agents apoptosis independent of the status of p53. Our results show that TSinhibitors inducing apoptosis may play a future potential role as new therapeutic agents formelanoma, but further studies are necessary to understand their molecular mechanism.