HCT116 (30 × 10^4 cells/well) were seeded into 24-well plates and cultured at standard conditions (37 °C, 5% CO2). After 24 h, the medium was replaced with fresh culture medium and cells were treated with a transfection mixture containing 0.75 μg of jetPRIME transfection reagent and 0.25 μg of pCI_AKR1C3 or empty pCI plasmid. The mixture was incubated for 10 min at room temperature and the polyplexes were added dropwise into the wells.

Members of the short-chain dehydrogenase/reductase (SDR) and aldo-keto reductase (AKR) superfamilies mediate the reduction of anthracyclines to their less potent C-13 alcohol metabolites. This reductive metabolism has been recognized as one of the most important factors that trigger anthracycline resistance in cancer cells. In our study, two purine analogues, purvalanol A and roscovitine, were identified as effective inhibitors of aldo-keto reductase 1C3 (AKR1C3), an enzyme that is overexpressed in many cancer types and is also a key player in tumour cell resistance to anthracyclines. Purvalanol A and roscovitine potently inhibited human recombinant AKR1C3 (Ki = 5.5 muM and 1.4 muM, respectively) and displayed similar activity in experiments with intact cells. Ligand-protein docking calculations suggested that both inhibitors occupied a part of the cofactor-binding site. Furthermore, we demonstrated that the combination of daunorubicin with purvalanol A or roscovitine exhibited a synergistic effect in AKR1C3 overexpressing cells. Based on these findings, it is possible to presume that purvalanol A and roscovitine may have the potential to enhance the therapeutic effectiveness and safety of anthracyclines via inhibition of AKR1C3.