The anthracycline doxorubicin is a front line antineoplastic agent with activity against a wide range of solid and blood tumours. Its mode of action is to stabilise topoisomerase-II-DNA cleavable complexes, inducing double stranded DNA breaks and leading to cell death (1). In the presence of formaldehyde this mechanism can be subverted to preferentially form covalent drug-DNA adducts, a mechanism that exerts a more potent effect in killing cancer cells in culture (2).
A panel of potential formaldehyde-releasing prodrugs (FRPs) (experimental compound AN-9, antibiotic Cefditoren Pivoxil and topoisomerase inhibitor Sobuzoxane) were tested in combination with doxorubicin against the aggressive metastatic 4T1.2 murine model of breast cancer. Single agent doxorubicin was used to induce topoisomerase II associated breaks (measured using the Comet assay) while combination with the formaldehyde-releasing prodrugs were used to induce DNA adducts (measured by covalent incorporation of 14C doxorubicin into DNA).
Doxorubicin and prodrug combinations reduced DNA breaks and promoted doxorubicin-DNA adduct formation consistent with a switch in mechanism of action of doxorubicin. Dox-FRP combinations also reduced cell growth to a greater extent than doxorubicin alone, whilst exhibiting limited toxicity on their own.
To elucidate cellular changes in response to the combination, transcriptomics analysis was performed using 4T1.2, EO771 (murine) and MDA-231 (human) breast cancer cell lines. Cells were treated for 24 h with 1µM of Doxorubicin with and without 100µM AN-9 and libraries were prepared for RNA sequencing. KEGG pathways associated with Spliceosome, RNA transport and ribosome biogenesis were upregulated in combination treated cells relative to other treatments.
In vivo studies indicate the combination of doxorubicin and AN-9 reduces expression of proliferation marker Ki-67 in primary tumours and reduces metastasis to lungs (as measured by luminescence of luciferase labelled cells).