Donald Ralph. Phillips

Formaldehyde-activated pixantrone is a monofunctional DNA alkylator that binds selectively to CpG and CpA doublets

The topoisomerase II poison mitoxantrone is important in the clinical management of human malignancies. Pixantrone, a novel aza-anthracenedione developed to improve the therapeutic profile of mitoxantrone, can efficiently alkylate DNA after formaldehyde activation. In vitro transcriptional analysis has now established that formaldehyde-activated pixantrone generates covalent adducts selectively at discrete CpG or CpA dinucleotides, suggesting that the activated complex binds to guanine or cytosine (or both) bases. The stability of pixantrone adduct-induced transcriptional blockages varied considerably, reflecting a mixture of distinct pixantrone adduct types that may include relatively labile monoadducts and more stable interstrand cross-links. 6,9-Bis-[[2-(dimethylamino)ethyl]amino]benzo[g]isoquinoline-5,10-dione (BBR 2378), the dimethyl N-substituted analog of pixantrone, could not form adducts, suggesting that pixantrone alkylates DNA through the primary amino functions located in each side chain of the drug. Pixantrone generated DNA adducts only when guanine was present in substrates and exhibited a lack of adduct formation with inosine-containing polynucleotides, confirming that the N2 amino group of guanine is the site for covalent attachment of the drug. Mass spectrometric analysis of oligonucleotide-drug complexes confirmed that formation of covalent pixantrone-DNA adducts is mediated by a single methylene linkage provided by formaldehyde and that this occurs only with guanine-containing double stranded oligonucleotide substrates. CpG methylation, an epigenetic modification of the mammalian genome, significantly enhanced the generation of pixantrone-DNA adducts within a methylated DNA substrate, indicating that the methylated dinucleotide may be a favored target in a cellular environment.

Formaldehyde-releasing prodrugs specifically affect cancer cells by depletion of intracellular glutathione and augmentation of reactive oxygen species

Histone deacetylase inhibitory prodrugs that are metabolized to carboxylic acid(s) and aldehyde(s) possess antineoplastic properties. Formaldehyde-releasing prodrugs were shown to be the most potent. The objective of this study was to gain understanding on the mode of action of these prodrugs in cancer cells. HL-60 and MCF-7 cells in the presence of N-acetylcysteine or glutathione were protected from death induced by formaldehyde-releasing prodrugs but not from death caused by the homologous acetaldehyde-releasing ones. Cell death induced by the former was accompanied by depletion of intracellular glutathione and increased reactive oxygen species that were attenuated by N-acetylcysteine. At fourfold higher concentration, acetaldehyde-releasing prodrugs increased reactive oxygen species that were further augmented by N-acetylcysteine. In HL-60 cells, formaldehyde-releasing prodrugs dissipated the mitochondrial membrane potential and glutathione or N-acetylcysteine restored it. Although acetaldehyde-releasing prodrugs dissipated mitochondrial membrane potential, it occurred at 20-fold greater concentration and was unaffected by the antioxidants. Formaldehyde-releasing prodrugs abrogated c-myc protein expression and elevated c-Jun and H2AX phosphorylation, N-acetylcysteine partially reversed these changes. Herein, we show that formaldehyde-releasing prodrugs diminish the level of glutathione most likely by forming S-formylglutathione adducts resulting in increase of reactive oxygen species followed by signaling events that lead to cancer cells death.

The cardio-protecting agent and topoisomerase II catalytic inhibitor sobuzoxane enhances doxorubicin-DNA adduct mediated cytotoxicity

PurposeThe importance of understanding the mechanism of action of anticancer agents is sometimes overlooked in the pursuit of new and therapeutically advantageous compounds. Doxorubicin has long been identified as an inhibitor of the DNA-decatenating enzyme topoisomerase II, this being believed to be the major mechanism of action of this drug. However, the complex nature of cytotoxicity induced by doxorubicin suggests that more than one mechanism of action is responsible for cell kill. Investigation into various other cellular effects has shown that doxorubicin can, in the presence of formaldehyde, form doxorubicin-DNA adducts, resulting in enhanced cell death.MethodsWe have used six catalytic inhibitors of topoisomerase II (aclarubicin, merbarone, suramin, staurosporine, maleimide and sobuzoxane) to investigate the role of topoisomerase II mediated cell effects in doxorubicin-DNA adduct inducing treatments. Adduct levels were determined by scintillation counting of [14C]doxorubicin-DNA lesions and DNA damage responses by Comet analysis and flow cytometry (apoptosis).ResultsHere we show that sobuzoxane inhibits topoisomerase II but in the presence of doxorubicin also enhances the production of doxorubicin-DNA adducts resulting in an enhanced cytotoxic response. We show that the formation of doxorubicin-DNA adducts is mediated by formaldehyde released from sobuzoxane when it is metabolised.ConclusionsSobuzoxane has also been shown to decrease the normally dose limiting cardiotoxicity commonly exhibited with clinical use of doxorubicin. The potential combination of doxorubicin and sobuzoxane in cancer chemotherapy has two advantages. First, the mechanism of doxorubicin toxicity is shifted away from topoisomerase II inhibition and towards drug-DNA adduct formation which may allow for a lower drug dose to be used and circumvent some drug resistance problems. Second, the addition of a cardioprotecting agent will counteract the commonly dose limiting side effect of cardiac damage resulting from doxorubicin treatment. The importance of the potentiation of cell kill of doxorubicin and sobuzoxane provides a rationalisation of a mechanistic-based combination of anticancer drugs for an improved clinical outcome.

Formaldehyde-releasing prodrugs in combination with Adriamycin can overcome cellular drug resistance.

The anticancer drug Adriamycin is widely used in cancer chemotherapy and is classified as a topoisomerase II inhibitor. However, in the presence of formaldehyde, Adriamycin also forms high levels of DNA adducts. In this study, a new series of butyric acid and formaldehyde-releasing drugs related to AN9 (pivaloyloxymethyl butyrate) was assessed for their ability to facilitate Adriamycin-DNA adduct formation in Adriamycin-sensitive and -resistant cell lines (HL60 and HL60/MX2; MES-SA and MES-SA/Dx5). Drugs that released two molar equivalents of formaldehyde per mole of prodrug were superior in their ability to enhance adduct formation compared to those that released one molar equivalent. Adduct formation (as assessed by binding of radiolabeled Adriamycin to genomic DNA) was always lower in the resistant cell lines compared to the sensitive cell lines. However, in growth inhibition experiments, prodrug combinations were able to overcome Adriamycin resistance to varying degrees, and the combination of Adriamycin with selected prodrugs that release two moles of formaldehyde totally overcame resistance in HL60/MX2 cells. These HL60-derived cells express altered levels of topoisomerase II and also express a mutant form of the enzyme. Combinations of Adriamycin with selected prodrugs that release one or two moles of formaldehyde partially overcame P-glycoprotein-mediated resistance in MES-SA/Dx5 cells. Formaldehyde-releasing prodrugs (as single agents) overcame both forms of resistance in the two resistant cell lines, demonstrating that they were not substrates of these resistance mechanisms. Collectively, these results suggest that changing the mechanism via which Adriamycin exerts its anticancer effect by dramatically increasing adduct levels (requiring coadministration of formaldehyde-releasing prodrugs) may be a useful means of cancer treatment, as well as for overcoming Adriamycin-induced resistance.

Processing of anthracycline-DNA adducts via DNA replication and interstrand crosslink repair pathways

Anthracycline chemotherapeutics are well characterised as poisons of topoisomerase II, however many anthracyclines, including doxorubicin, are also capable of forming drug-DNA adducts. Anthracycline-DNA adducts present an unusual obstacle for cells as they are covalently attached to one DNA strand and stabilised by hydrogen bonding to the other strand. We now show that in cycling cells processing of anthracycline adducts through DNA replication appears dominant compared to processing via transcription-coupled pathways, and that the processing of these adducts into DNA breaks is independent of topoisomerase II. It has previously been shown that cells deficient in homologous recombination (HR) are hypersensitive to adduct forming treatments. Given that anthracycline-DNA adducts, whilst not true crosslinks, are associated with both DNA strands, the role of ICL repair pathways was investigated. Mus81 is a structure specific nuclease implicated in Holliday junction resolution and the resolution of branched DNA formed by stalled replication forks. We now show that ICL repair deficient cells (Mus81(-/-)) are hypersensitive to anthracycline-DNA adducts and ET-743, a compound which causes a chemically similar type of DNA damage. Further analysis of this mechanism showed that Mus81 does not appear to cause DNA breaks resulting from either anthracycline- or ET743-DNA adducts. This suggests Mus81 processes these novel forms of DNA damage in a fundamentally different way compared to the processing of classical covalent crosslinks. Improved understanding of the role of DNA repair in response to such adducts may lead to more effective chemotherapy for patients with BRCA1/2 mutations and other HR deficiencies.

Abstract C279: Novel anthracenedione derivatives demonstrating several potential modes of actions.

DNA has been regarded as the primary target of numerous non-specific cytotoxic anticancer agents such as the nitrogen mustards and anthracycline antibiotics. The anthracyclines, most notably doxorubicin, proved to be highly potent and were thus incorporated into numerous chemotherapeutic regimens. However, their dose dependent redox activity that presumably contributes to their broad spectrum versatility is also associated with potentially lethal cardiotoxic adverse effects and thus, inspired the development of synthetic analogues including the anthracenediones. Like the anthracylines, their structural frame constitutes the DNA intercalating anthraquinone chromophore with functional amino groups stabilizing its insertion via hydrogen bonds and Van der Waal interactions. This interaction poisons the topoisomerase II cleavable complex which induces widespread DNA strand breaks culminating in the induction of apoptosis. We have shown that the availability of the methyl donating molecule, formaldehyde, generates adduct formation via a covalent aminal linkage between the exocyclic N2 amino group of guanine residue at specific CpG dinucleotides and an amino group on the anthracenediones side chains. The formaldehyde mediated DNA adduct formation, achievable via simultaneous formaldehyde-releasing prodrug administration, increases cancer cell toxicity and potentially endows general cardioprotective activity. Such cardioprotection has been associated with formaldehyde-mediated doxorubicin-DNA adducts We have synthesised a new series of anthracenediones that vary in their potential to induce covalent DNA adducts. We now show that the new anthracenediones including pixantrone (unlike the anthracyclines) also exhibits a preference for forming adducts with single stranded DNA and RNA, and these have similar characteristics to adducts that are formed with DNA. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C279. Citation Format: Paul P. Pumuye, Jelena E. Medan, Brad Sleebs, Keith Watson, Benny J. Evison, Donald R. Phillips, Suzanne M. Cutts. Novel anthracenedione derivatives demonstrating several potential modes of actions. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C279.