Nuclear targeting and nuclear retention of anthracycline-formaldehyde conjugates implicates DNA covalent bonding in the cytotoxic mechanism of anthracyclines

Abstract

The anthracycline, antitumor drugs doxorubicin (DOX), daunorubicin (DAU), and epidoxorubicin (EPI) catalyze production of formaldehyde through induction of oxidative stress. The formaldehyde then mediates covalent bonding of the drugs to DNA. Synthetic formaldehyde conjugates of DOX, DAU, and EPI, denoted Doxoform (DOXF), Daunoform (DAUF), and Epidoxoform (EPIF), exhibit enhanced toxicity to anthracycline-sensitive and -resistant tumor cells. Uptake and retention of parent anthracycline antitumor drugs (DOX, DAU, and EPI) relative to those of their formaldehyde conjugates (DOXF, DAUF, and EPIF) were assessed by flow cytometry in both drug-sensitive MCF-7 cells and drug-resistant MCF-7/ADR cells. The MCF-7 cells took up more than twice as much drug as the MCF-7/ADR cells, and both cell lines took up substantially more of the formaldehyde conjugates than the parent drugs. Both MCF-7 and MCF-7/ADR cells retained fluorophore from DOXF, DAUF, and EPIF hours after drug removal, while both cell lines almost completely expelled DOX, DAU, and EPI within 1 h. Longer treatment with DOX, DAU, and EPI resulted in modest drug retention in MCF-7 cells following drug removal but poor retention of DOX, DAU, and EPI in MCF-7/ADR cells. Fluorescence microscopy showed that the formaldehyde conjugates targeted the nuclei of both sensitive and resistant cells, and remained in the nucleus hours after drug removal. Experiments in which [(3)H]Doxoform was used, synthesized from doxorubicin and [(3)H]formaldehyde, also indicated that Doxoform targeted the nucleus. Elevated levels of (3)H were observed in DNA isolated from [(3)H]Doxoform-treated MCF-7 and MCF-7/ADR cells relative to controls. The results implicate drug-DNA covalent bonding in the tumor cell toxicity mechanism of these anthracyclines.