In vitro evaluation of fotemustine as a potential agent for limb perfusion in melanoma

The mechanism of action of fotemustine, a relatively new chloroethylnitrosourea, was evaluated in human melanoma cells in order to assess its potential as an agent for hyperthermic limb perfusion. Fotemustine was more toxic to O6-alkylguanine methyl transferase (AGT) deficient (Mer-) cells than Mer+ cells, implicating AGT as a major determinant of resistance. Mer+ cells derived from Mer- cell lines following exposure to the monofunctional alkylating metabolite of dacarbazine (DTIC) were also resistant to fotemustine. Mer status did not influence the replication of fotemustine-damaged adenovirus 5, whereas virus treated with the monofunctional alkylating agent N-methyl-N1-nitro-N-nitrosoguanidine (MNNG) was replicated much more efficiently by Mer+ cells. This suggests that the initial O6-alkylated product, if not immediately repaired, rearranges to form DNA crosslinks which cannot be repaired by AGT. Replication of a control virus was not affected by treating the cells with fotemustine, indicating that the drug acted primarily on DNA rather than at epigenetic levels. Fotemustine generally produced a G2-M block in the cell cycle, most strikingly in Mer- cells at low, minimally toxic concentrations; MNNG and high doses of fotemustine induced S phase arrest. Concurrent hyperthermia (41.5 degrees C for 1 h) increased the toxicity of fotemustine in some cell lines. Fotemustine decomposed in culture medium in two phases; the first was complete within 5 min and was most marked in Mer+ cells. The results suggest that fotemustine may be suitable for isolated limb perfusion in melanoma, with the potential for overcoming resistance by including inhibitors of AGT.