Activation of clinically used anthracyclines by the formaldehyde-releasing prodrug pivaloyloxymethyl butyrate.

The anthracycline group of compounds is extensively used in current cancer chemotherapy regimens and is classified as topoisomerase II inhibitor. However, previous work has shown that doxorubicin can be activated to form DNA adducts in the presence of formaldehyde-releasing prodrugs and that this leads to apoptosis independently of topoisomerase II-mediated damage. To determine which anthracyclines would be useful in combination with formaldehyde-releasing prodrugs, a series of clinically relevant anthracyclines (doxorubicin, daunorubicin, idarubicin, and epirubicin) were examined for their capacity to form DNA adducts in MCF7 and MCF7/Dx (P-glycoprotein overexpressing) cells in the presence of the formaldehyde-releasing drug pivaloyloxymethyl butyrate (AN-9). All anthracyclines, with the exception of epirubicin, efficiently yielded adducts in both sensitive and resistant cell lines, and levels of adducts were similar in mitochondrial and nuclear genomes. Idarubicin was the most active compound in both sensitive and resistant cell lines, whereas adducts formed by doxorubicin and daunorubicin were consistently lower in the resistant compared with sensitive cells. The adducts formed by doxorubicin, daunorubicin, and idarubicin showed the same DNA sequence specificity in sensitive and resistant cells as assessed by lambda-exonuclease-based sequencing of alpha-satellite DNA extracted from drug-treated cells. Growth inhibition assays were used to show that doxorubicin, daunorubicin, and idarubicin were all synergistic in combination with AN-9, whereas the combination of epirubicin with AN-9 was additive. Although apoptosis assays indicated a greater than additive effect for epirubicin/AN-9 combinations, this effect was much more pronounced for doxorubicin/AN-9 combinations.

The hydroxyl epimer of doxorubicin controls the rate of formation of cytotoxic anthracycline-DNA adducts.

Epirubicin was developed as a semi-synthetic anthracycline derivative to circumvent the cardiotoxic limitations associated with the use of doxorubicin in the clinic. Anthracycline compounds have been demonstrated to form covalent drug-DNA adducts utilising endogenous and exogenous sources of formaldehyde; however, previous investigations of the formation of epirubicin-DNA adducts provide conflicting evidence for adduct formation. This work provides evidence that epirubicin acts to form drug-DNA adducts at physiologically relevant concentrations and demonstrates that the rate of formation of epirubicin-DNA adducts is slower than that observed for other anthracycline compounds, explaining why they are only detectable under defined experimental conditions. Formation of covalent epirubicin-DNA adducts improves the apoptotic profile of epirubicin and provides opportunities to overcome drug resistance and cardiotoxic limitations.

Activation of DNA damage response pathways as a consequence of anthracycline-DNA adduct formation.

The cytotoxicity of doxorubicin, a clinically used anti-neoplastic drug, can be enhanced by formaldehyde (either endogenous or exogenous) to promote the formation of doxorubicin-DNA adducts. Formaldehyde supplies the carbon required for the covalent linkage of doxorubicin to one strand of DNA, with hydrogen bonds stabilising the doxorubicin mono-adduct to the other strand of DNA, to act much like an interstrand crosslink. Interstrand crosslinks present a major challenge for cellular repair processes, requiring the activation of numerous DNA damage response proteins for resolution of the resulting DNA intermediates and damage. This work investigates DNA damage response proteins activated by doxorubicin-DNA adducts. Although p53 was phosphorylated at Serine 15 in response to adducts, long term growth inhibition of mammalian cells was not affected by p53 status. Using siRNA technology and kinase inhibitors we observed enhanced cellular sensitivity to doxorubicin-DNA adducts when the activity of the signalling protein kinases ATM and ATR were lost. Cells synchronised using a double thymidine block were sensitised to adduct-initiated cell death upon ATR knockdown, but relatively unaffected by ATM knockdown. Loss of ATR was associated with abrogation of a drug-induced G(2)/M block and induction of mitotic catastrophe, while loss of ATM was associated with drug-induced apoptosis in non-synchronised cells. These proteins may therefore be potential drug targets to achieve synergistic cytotoxic responses to doxorubicin-DNA adduct forming therapies. The analysis of these protein kinases with respect to cell cycle progression indicates that ATR is required for G(2)/M checkpoint responses while ATM appears to function in G(1) mediated responses to anthracycline adducts.

M2, a novel anthracenedione, elicits a potent DNA damage response that can be subverted through checkpoint kinase inhibition to generate mitotic catastrophe.

Pixantrone is a promising anti-cancer aza-anthracenedione that has prompted the development of new anthracenediones incorporating symmetrical side-chains of increasing length varying from two to five methylene units in each pair of drug side-chains. A striking relationship has emerged in which anthracenedione-induced growth inhibition and apoptosis was inversely associated with side-chain length, a relationship that was attributable to a differential ability to stabilise the topoisomerase II (TOP2) cleavage complex. Processing of the complex to a DNA double strand break (DSB) flanked by γH2AX in nuclear foci is likely to occur, as the generation of the primary lesion was antecedent to γH2AX induction. M2, bearing the shortest pair of side-chains, induced TOP2-mediated DSBs efficiently and activated cell cycle checkpoints via Chk1 and Chk2 phosphorylation, implicating the involvement of ATM and ATR, and induced a protracted S phase and subsequent G2/M arrest. The inactive analogue M5, containing the longest pair of side-chains, only weakly stimulated any of these responses, suggesting that efficient stabilisation of the TOP2 cleavage complex was crucial for eliciting a strong DNA damage response (DDR). An M2 induced DDR in p53-defective MDA-MB-231 cells was abrogated by UCN-01, a ubiquitous inhibitor of kinases including Chk1, in a response associated with substantial mitotic catastrophe and strong synergy. The rational selection of checkpoint kinase inhibitors may significantly enhance the therapeutic benefit of anthracenediones that efficiently stabilise the TOP2 cleavage complex.