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.

Figure 1.. Viability of RKO cells exposed to DAB.

RKO cells are seeded in 96-well plates at a density of 8,000 cells/well in DMEM supplemented with 10% FBS 24 h prior to DAB treatment. All DAB assays are performed in phenol red-free OptiMEM-reduced serum medium. Cell viability is assessed by WST-1 reduction levels compared to controls. Cell viability at different DAB concentrations after 5 or 24 h of incubation (A). IC ₅₀ calculation from the cell viability of RKO cells after 24 h of treatment (B).

Figure 2.. Effect of DAB on intra/extracellular redox status in RKO cells.

RKO cells are seeded in 96-well plates at 12,000 cells/well in DMEM supplemented with 10% FBS 24 h prior to each treatment. Cells are pre-loaded with fresh 30μM DCFH-DA in phenol red-free OptiMEM-reduced serum medium for 60 min at 37°C. The cells are subsequently rinsed thrice with PBS at RT and treated with different concentrations of DAB or H₂O₂. The fluorescence intensities are read at 488/530 nm immediately after the addition of DAB or H at the presented times (A). RKO cells are seeded in 24-well plate at 5 × 10⁴ cells/well in DMEM supplemented with 10% FBS 24 h prior to the assay. The cells are incubated with different DAB concentrations in presence of 0.1% NBT prepared in phenol red free-MEM, supplemented with 2% FBS for 3 h at 37°C. The formazan product in the medium is measured at 560 nm. A control assay is performed with 0.3 mM DAB in presence of 50 U CuZnSOD (B).

Figure 3.. Levels of thiol groups and role of GSH in RKO cells under DAB treatment.

RKO cells (3.5 × 10⁵) are treated with DAB or H₂O₂ for 5 h in OptiMEM. The total thiol groups are measured in TCA-precipitate, protein-SH and soluble fractions, and low molecular weight thiols. The thiol groups are accessed by reactions with monobromobimane (A). RKO cells (5 × 10⁴) are pretreated with NAC or BSO in phenol red free-MEM, supplemented with 2% FBS for 24 h at 37°C. Subsequently, cells are treated with 0.3 mM DAB and after 24 h the viability was evaluated (B). RKO cells (3.5 × 10⁵) are treated at the same conditions and the levels of GSH are measured by the enzymatic recycling method [23] (C).

Figure 4.. Effects of antioxidants on DAB toxicity to RKO cell line.

RKO cells are seeded in 24-well plates at 5 × 10⁴ cells/well in DMEM supplemented with 10% FBS 24 h prior to treatments. The cells are treated with DAB in medium supplemented with 5 μM catalase, 20 mM aminoguanidine, 5 mM NAC or 5 mM GSH for 24 h in phenol red free-MEM, supplemented with 2% FBS.

Figure 5.. Effect of the cyclic end products of DAB oxidation (oxoDAB derivatives) on RKO viability.

RKO cells are seeded in 24-well plates at 5 × 10⁴ cells/well in DMEM supplemented with 10% FBS 24 h prior to treatments. DAB (10 mM in PBS) is aged at 37°C for 24 h, in presence or absence of 20 mM of aminoguanidine, or NAC or GSH. The cells are treated with 1 mM of DAB-aged solution in phenol red free-MEM, supplemented with 2% FBS for 24 h (A). UV-Vis spectra of 24 h aged solutions of DAB in PBS in the presence and absence of 20 mM aminoguanidine, NAC or GSH. Spectra obtained from the DAB-NAC (dotted line) and DAB-GSH (dot-dashed line) systems are similar (B).

Figure 6.. Effect of DAB on the expression of HO-1 and other antioxidant response factors.

RKO cells are seeded in 100 mm dish at a density of 2 × 10⁶ cells in DMEM supplemented with 10% FBS 24 h prior to DAB treatment. RKO cells are treated with different concentrations of DAB during 5 or 24 h in phenol red-free OptiMEM-reduced serum medium. The cultures are maintained in a humidified atmosphere of 5% CO and 95% air at 37°C. Protein and RNA samples are submitted for to Western blot analysis (A) and RT-PCR analysis (B and C), respectively.

Figure 7.. Effects of DAB on the apoptotic pathways of RKO cell lineage.

RKO cells are seeded in a 100 mm dish at a density of 2 × 10⁶ cells in DMEM supplemented with 10% FBS 24 h prior to DAB treatment. RKO cells are treated with different concentrations of DAB for 24 h in phenol red-free OptiMEM-reduced serum medium. Protein samples are submitted to Western blot analysis using specific primary antibodies (A). The effect of pan-caspase inhibitors on DAB toxicity is evaluated by a cell viability assay using the WST-1 reagent, in which RKO cells are seeded at 8,000 cells/well in 96-well plates in DMEM supplemented with 10% FBS 24 h before each treatment. The cells are previously treated with 10 μM z-VAD-FMK, followed by DAB treatment for 24 h in phenol red-free OptiMEM-reduced serum medium (B).

Figure 8.. Effect of DAB treatment on cell cycle and related proteins.

RKO cells are treated with 0.30 mM DAB for 24 h in OptiMEM. After time-lapse incubation, the cell suspensions (2 × 10⁶ cells/ml) are fixed in cold 70% ethanol overnight at 4°C, washed with PBS and stained with 50 μ g/mL iodide propidium solution in DPBS containing 50 μg/mL of RNase A for 30 min. The samples are analyzed by a flow cytometer (A). Protein samples are submitted to Western blotting analysis in which specific primary antibodies are used (B).

Figure 9.. Envisaged mechanism to explain the cytotoxic effects of DAB on mammalian cells.

DAB undergoes metal-catalyzed aerobic oxidation yielding oxyradicals and an α-oxoaldehyde at both extra and intracellular environments. Increased ROS levels changes the cell redox balance leading to depletion of protective thiols. The resulting oxidizing triggers up-regulated stress response pathways such as Nrf-2, HO-1, NQO1 and xCT, as well as leading to activation of caspases cascades, PARP cleavage and subsequently apoptosis.