Mechanism of Action of Fotemustine, a New Chloroethylnitrosourea Anticancer Agent:  Evidence for the Formation of Two DNA-Reactive Intermediates Contributing to Cytotoxicity

Abstract

Methyl excision repair deficient human tumor cells (Mer-) were found to be hypersusceptible to killing by the antimelanoma agent fotemustine (FM) implicating alkylation of O6 guanine as the major contributor to toxicity. Preincubation of the drug in aqueous solution for 5 min resulted in an immediate reduction in cytotoxicity (35-50%), in vitro DNA alkylation (31%), and DNA interstrand cross-linking (40%) followed by a second reaction with considerably slower kinetics. Electrospray ionisation mass spectrometry (ESI-MS) showed that in aqueous solution FM rearranged rapidly to form either a metastable tautomer or decomposed to form a highly reactive diazohydroxide (t1/2 < 2 min). These results suggest the presence of two DNA-reactive species relevant to biological activity. Coincubation of ellagic acid (an inhibitor of O6-guanine alkylation) with FM inhibited in vitro ISC, suggesting that the O6-chloroethyl lesion is the predominant cause of the cross-link. On the basis of these findings, we propose that FM breaks down to form a short-lived intermediate, 2-chloroethyldiazohydroxide, which rapidly generates O6-guanine lesions responsible for the drug's initial activity and a long lived iminol tautomer responsible for the remaining O6 guanine alkylation and cytotoxicity.

Treatment of disseminated ocular melanoma with sequential fotemustine, interferon α, and interleukin 2

“…thioredoxin reductase, glutathione reductase, and ribonucleotide reductase, resulting in their inhibition. Moreover, it has alkylating and carbamoylating effects on nucleic acids (Hayes et al, 1997). Both effects may induce the expression of stress molecules such as hsp70 or MICA which can link chemo-and immunotherapy (Weichenthal et al, 1998).…”

Electrospray ionization mass spectrometry of oligonucleotide complexes with drugs, metals, and proteins

“…The activation of tamoxifen, which is widely used for the treatment of breast cancer, has been studied in rats, mice, and humans using 32 P-postlabeling and LC-MS (Phillips et al, 1996). ESI-MS has been used to study the mode of action of fotemustine, a chloroethylnitrosourea antitumor agent, that alkylates guanine (Hayes et al, 1997). Finally, a number of mitosene±DNA adducts have also been detected by ESI-MS in combination with other techniques (Maliepaard et al, 1997).…”

Assessment of DNA interstrand crosslinks in NIH/3T3 cells induced by Chloroethylnitrosoureas

“…They damage DNA by alkylation of bases, single/double strand breakages and interstrand crosslinks [14][15]. DNA damage caused by different nitrosoureas has been studied via different methods of detection, such as agarose gel electrophoresis assay [16], fluorescence assay [17] and lipid chromatography/mass spectrometry (LC/MS) [18]. Being highly sensitive and specific, the comet assay proved to be a useful technique to evaluate the degree of DNA damage [19].…”