Abstract
Chloroethylnitrosoureas (CENUs), which are bifunctional alkylating agents widely used in the clinical treatment of cancer, exert anticancer activity by inducing crosslink within a guanine-cytosine DNA base pair. However, the formation of dG-dC crosslinks can be prevented by O6-alkylguanine-DNA alkyltransferase (AGT), ultimately leading to drug resistance. Therefore, the level of AGT expression is related to the formation of dG-dC crosslinks and the sensitivity of cells to CENUs. In this work, we determined the CENU-induced dG-dC crosslink in mouse L1210 leukemia cells and in human glioblastoma cells (SF-763, SF-767 and SF-126) containing different levels of AGT using high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The results indicate that nimustine (ACNU) induced more dG-dC crosslinks in L1210 leukemia cells than those induced by carmustine (BCNU), lomustine (CCNU) and fotemustine (FTMS). This result was consistent with a previously reported cohort study, which demonstrated that ACNU had a better survival gain than BCNU, CCNU and FTMS for patients with high-grade glioma. Moreover, we compared the crosslinking levels and the cytotoxicity in SF-763, SF-767 and SF-126 cells with different AGT expression levels after exposure to ACNU. The levels of dG-dC crosslink in SF-126 cells (low AGT expression) were significantly higher than those in SF-767 (medium AGT expression) and SF-763 (high AGT expression) cells at each time point. Correspondingly, the cytotoxicity of SF-126 was the highest followed by SF-767 and SF-763. The results obtained in this work provided unequivocal evidence for drug resistance to CENUs induced by AGT-mediated repair of DNA ICLs. We postulate that the level of dG-dC crosslink has the potential to be employed as a biomarker for estimating drug resistance and anticancer efficiencies of novel CENU chemotherapies.
Highlights
Chloroethylnitrosoureas (CENUs) are bifunctional anti-tumor alkylating agents that have important clinical applications for the treatment of cancer, such as lymphomas, melanomas, and cerebromas [1,2,3]
The method was used for quantifying the dG-dC crosslinks in human glioma SF-126, SF-763 and SF-767 cells treated with ACNU and in L1210
Quantitative analyses were performed to determine the dG-dC crosslinks induced by CENUs in mouse L1210 leukemia cells and human SF-126, SF-763 and SF-767 glioma cells using HPLC-ESI-MS/MS
Summary
Chloroethylnitrosoureas (CENUs) are bifunctional anti-tumor alkylating agents that have important clinical applications for the treatment of cancer, such as lymphomas, melanomas, and cerebromas [1,2,3]. We reported an HPLC-MS/MS quantification of dG-dC crosslink induced by ACNU, BCNU, CCNU and FTMS in SF-763, SF-767, SF-126 and L1210 cells containing different levels of AGT. For the groups treated with BCNU, CCNU and FTMS, the maximal crosslinking levels were 1243, 783 and 1387 fmol/mg DNA, respectively, which were observed at 12 h with the highest drug concentration.
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