Cytotoxicity, DNA Damage, and Apoptosis Induced by New Fotemustine Analogs on Human Melanoma Cells in Relation to O⁶-Methylguanine DNA-Methyltransferase Expression

Abstract

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 O 6 -alkyl groups by the enzyme O 6 -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 MerϪ melanoma cells and of MGMT inhibition with O 6 -benzylguanine (BG) in A375 Merϩ 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 O 6 -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. O 6 -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.

Acquired Resistance to Temozolomide in Glioma Cell Lines: Molecular Mechanisms and Potential Translational Applications

“…O6 -BG (5 µ M ) was used to try to deplete MGMT in the MGMT-transfected glioma lines; however, neither SNB19M nor U373M cells were sensitized to TMZ by O6 -BG, even when concentrations of O6 -BG were raised to 100 µ M . These cells continuously produce extremely high levels of MGMT protein, and sufficient concentrations of O6 -BG could not be achieved to deplete transfected cells of enzyme activity [30]. …”

Temozolomide- and fotemustine-induced apoptosis in human malignant melanoma cells: response related to MGMT, MMR, DSBs, and p53

“…In a recent study it was reported that TMZ does not induce apoptosis, but senescence in human melanoma cells (Mhaidat et al, 2007), which is surprising in view of the fact that other cell systems such as malignant gliomas undergo apoptosis very efficiently in response to TMZ (Roos et al, 2007a). For fotemustine, it has been shown that melanoma cells are killed by apoptosis (Passagne et al, 2003;Kissel et al, 2006), but detailed studies on the mode of cell death by this drug in melanoma cells are not available. Also, there is no systematic study (utilising the same cell model) for melanomas as to the role of MGMT, p53, and MMR proteins in the O 6 -alkylguanine-triggered killing response.…”

Anti-Tumour Activity of Fotemustine and Protons in Combination with Bevacizumab

“…These cells are among the very radio-resistant in melanomas with a surviving fraction of 0.93 at 2 Gy protons [18] . FM has been shown to have a significant inhibitory effect on other melanoma cell lines in vitro [27,28] . This drug has increased the radio-sensitivity of HTB140 melanoma cells [29] .…”

“…Irradiation doses of 12 and 16 Gy are in the range of those used in proton therapy [18] . The time point of 48 h after treatment was appropriate for the analysis of the drug effects as well as for the evaluation of the pro-apoptotic ability of the different treatments [28,36] . FM has shown the best inhibitory effects on HTB140 melanoma cell viability and proliferation between 48 and 72 h [32] .…”