Fotemustine is a third generation chloroethylnitrosourea that has demonstrated significant antitumoral effects in
malignant melanoma. However, its use is somewhat limited by its toxic side effects and chemoresistance caused by direct repair of O6-alkyl groups by the
enzyme O6-methylguanine DNA-
methyltransferase (MGMT). The aim of this work was to determine to what extent the expression of MGMT influences cytotoxicity, DNA damage, and apoptosis induced by new nitrososulfamide analogs of
fotemustine (compounds 4 and 8), which have previously demonstrated interesting antiproliferative properties. We carried out complementary strategies that consisted of MGMT
cDNA transfection in CAL77 Mer-
melanoma cells and of MGMT inhibition with
O6-benzylguanine (BG) in A375 Mer+
melanoma cells. MGMT-transfected cells were 7 to 9 times less sensitive to
fotemustine than parent cells, whereas no difference between the transfected and parent cells was observed for nitrososulfamide analogs. The cytotoxicity of these analogs vis à vis a MGMT-proficient A375
melanoma cell line was approximately 3 times greater than that of
fotemustine. Coincubation of these cells with
O6-benzylguanine significantly increased the cytotoxicity of
fotemustine and compound 8, whereas BG had little effect on the cytotoxicity of compound 4. Furthermore, DNA fragmentation determined by a comet assay was greater with nitrososulfamide analogs than with
fotemustine.
O6-benzylguanine increased DNA fragmentation for
fotemustine and compound 8, but not for compound 4, which induced comets with a typical apoptotic appearance. The ability of this compound to induce apoptosis in the absence of BG was confirmed by a specific
enzyme-linked
immunosorbent assay apoptotic assay using a
single-stranded DNA monoclonal antibody.