Cytotoxicity of 1,4-diamino-2-butanone, a putrescine analogue, to RKO cells: mechanism and redox imbalance

Abstract

alpha-Aminocarbonyl metabolites (e.g., 5-aminolevulinic acid and aminoacetone) and the wide spectrum microbicide 1,4-diamino-2-butanone (DAB) have been shown to exhibit pro-oxidant properties. in vitro, these compounds undergo phosphate-catalyzed enolization at physiological pH and subsequent superoxide radical-propagated aerobic oxidation, yielding a reactive alpha-oxoaldehyde and H2O2. DAB cytotoxicity to pathogenic microorganisms has been attributed to the inhibition of polyamine biosynthesis. However, the role played in cell death by reactive DAB oxidation products is still poorly understood. This work aims to clarify the mechanism of DAB-promoted pro-oxidant action on mammalian cells. DAB (0.05-10 mM) treatment of RKO cells derived from human colon carcinoma led to a decrease in cell viability (IC50 ca. 0.3 mM DAB, 24 h incubation). Pre-addition of either catalase (5 mu M) or aminoguanidine (20 mM) was observed to partially inhibit the toxic effects of DAB to the cells, while N-acetyl-L-cysteine (NAC, 5 mM) or reduced glutathione (GSH, 5 mM) provided almost complete protection against DAB. Changes in redox balance and stress response pathways were indicated by the increased expression of HO-1, NQO1 and xCT. Moreover, the observation of caspase 3 and PARP cleavage products is consistent with DAB-triggered apoptosis in RKO cells, which was corroborated by the partial protection afforded by the pan-caspase inhibitor z-VAD-FMK. Finally, DAB treatment disrupted the cell cycle in response to increased p53 and activation of ATM. Altogether, these data support the hypothesis that DAB exerts cytotoxicity via a mechanism involving not only polyamine biosynthesis but also by DAB oxidation products.