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

Abstract

α-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 α-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 μM) 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.

Keywords::

• 1,4-diamino-2-butanone
• redox imbalance
• RKO cells
• apoptosis
• hydrogen peroxide
• α-oxoaldehyde

Acknowledgments

We are grateful to Dr. Maria Julia Manso Alves (IQUSP, Brazil) for helpful discussions and kindly reading this manuscript.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), INCT Processos Redox em Biomedicina – Redoxoma, and the National Institute of Environmental Health Sciences (NIEHS grant 5P01ES013125).