Cytotoxicity-related effects of imidazolium and chlorinated bispyridinium oximes in SH-SY5Y cells

Figure 1 shows the structures of the tested chlorinated bispyridinium oximes K867 and K870 and imidazolium oximes VII and X, prepared as described elsewhere (15, 16, 17) and donated to us by Dr Kamil Musilek (University of Hradec Králové Faculty of Science, Department of Chemistry, Hradec Králové, Czech Republic) and Dr Ines Primožič (University of Zagreb Faculty of Science, Department of Chemistry, Zagreb, Croatia).

Figure 1

The human neuroblastoma SH-SY5Y (ECACC 94030304) cell line was purchased from Sigma-Aldrich (Steinheim, Germany), the certified distributor for European Collection of Authenticated Cell Cultures (ECACC), and the cells were grown in Dulbecco’s Modified Eagle Medium (DMEM/F-12, Sigma-Aldrich) supplemented with 15 % (v/v) foetal bovine serum (Sigma-Aldrich), 2 mmol/L glutamine, and 1 % (v/v) non-essential amino acids (NEAA, Sigma-Aldrich) at 37 °C in a 5 % CO₂ atmosphere.

Cell viability was determined based on quantification of adenosine triphosphate (ATP) using the CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Madison, WI, USA), as the quantity of ATP is directly proportional to the number of metabolically active cells. 20,000 cells per well were seeded in 96-well opaque plates and exposed for 4 h to oximes in concentrations causing 20–25 % inhibition (~IC_20–25) as determined in our previous study (15). These were the following: 400 μmol/L for K867, 15 μmol/L for K870, 250 μmol/L for VII, and 200 μmol/L for X. After the incubation with the oximes, we measured cell luminescence on a VICTOR³ Multilabel Plate Reader (PerkinElmer, Waltham, MA, USA). Data were taken from at least two independent experiments (each treatment performed in duplicate) and plotted as a percentage of ATP determined in control, untreated cells.

SH-SY5Y cell preparation and the procedure followed a protocol described elsewhere in detail (18, 19). Briefly, the cells were seeded at a density of 100,000 cells per well in a complete medium with all supplements added. The following day, the medium was replaced with DMEM/F-12 without supplements. The cells were serum-starved for 16 h. During the last 5, 15, 30, 45, and 60 min, the cells were treated with the oximes in concentrations mentioned above. At the end of the experiment, cells were washed with ice-cold phosphate-buffered saline (PBS) to remove oximes and lysed in the Laemmli buffer containing 62.5 mmol/L Tris-HCl (pH 6.8), 2 % (w/v) sodium dodecyl sulphate (SDS), 10 % (w/v) glycerol, 5 % (v/v) 2-mercaptoethanol, and 0.002 % (w/v) bromophenol blue. Proteins were separated with sodium dodecyl sulphate and polyacrylamide gel (4–12 %) electrophoresis (SDS-PAGE) (Bio-Rad Laboratories, Hercules, CA, USA) and transferred to the polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt, Germany) by wet electrotransfer. After blocking, membranes were incubated overnight with a primary antibody (Table 1) diluted in a buffer containing 20 mmol/L Tris, 150 mmol/L NaCl, pH 7.5, 0.1 % (w/v) bovine serum albumin, and 0.1% (w/v) sodium azide at 4 °C and then with the secondary antibody-horseradish peroxidase conjugate in Tris-buffered saline with 0.1 % Tween 20 (TBST) and 5 % (w/v) dry milk at room temperature for 1 h. Membranes were incubated with an enhanced chemiluminescence (ECL) reagent (Bio-Rad Laboratories) and immunolabelled proteins (chemiluminiscent signal) visualised with a Fusion FX System (Vilber, Marne-la-Vallée, France). Densitometry was done with the Quantity One 1-D Analysis Software (Bio-Rad Laboratories). The intensities of individual bands are expressed in arbitrary units (AU) relative to the total intensity of all the bands. The Ponceau staining was used as protein loading/concentration control as described elsewhere (20).

Intracellular calcium (iCa²⁺) mobilised by the activation of G-protein coupled receptors (GPCR) or calcium channels was measured using the acetoxy-methyl-ester Fura-2 (Fura-2 AM) dye (Sigma-Aldrich). 30,000 cells per well were seeded in 96-well opaque plates, added the Fura-2 AM dye loading solution (100 μL/well), and incubated at room temperature for 1 h for the dye to penetrate the cells and get activated by cellular esterases. After washing cells twice with HEPES-buffered saline (HBS) containing 145 mmol/L NaCl, 5 mmol/L KCl, 1 mmol/L CaCl₂, 1 mmol/L MgCl₂, 10 mmol/L HEPES, and 10 mmol/L glucose (pH 7.4), cells were exposed to ~IC_20–25 concentrations of oximes reported in our previous study (15). Each well was monitored for baseline fluorescence, and, 25 s later, added 5 μmol/L of acetylcholine (ACh) (Sigma-Aldrich) using the automated pipetting system of an InfiniteM200PRO plate reader (Tecan Austria GmbH, Salzburg, Austria). Fluorescence was read at Ex/Em 340/510 nm for calcium-bound and at Ex/Em 380/510 nm for unbound Fura-2 dye, and the data are presented as ratiometric levels of the dye (340/380 nm) (21) indicating cytosolic calcium levels.

For target prediction in silico we used the online SwissTargetPredicition web tool (Swiss Institute of Bioinformatics, Lausanne, Switzerland), which can predict the most probable target proteins of small bioactive molecules from a library of 370,000 known active molecules and more than 3,000 proteins from three species (22, 23).

If not stated otherwise, results are presented as means ± standard error. Differences between the groups were analysed using one-way ANOVA followed by Dunnett’s test. Statistical analyses were run on the GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance is set as follows: &p≤0.05; #p≤0.01; $p≤0.001; and *p≤0.0001.

Unlike K867 and K870, oximes VII and X significantly decreased ATP (Figure 2).

Figure 2

Figure 3 shows time-dependent effects of the four oximes on important signalling kinases, transcription factors, and nuclear factors or enzymes regulating cell metabolism, growth, proliferation, and survival. The phosphorylation of extracellular signal-regulated kinase (ERK1/2), an important mitogen-activated protein kinase (p44/42 MAPK), increased with the chlorinated bispyridinium oximes K867 and K870 and decreased with the imidazolium oximes VII and X. The nuclear factor-κB (NF-κB), a transcription factor involved in the immune and inflammatory responses, showed no change in phosphorylation. K870 and X increased phosphorylation of p38 MAPK. Chlorinated bispyridinium oximes suppressed phosphorylation of the signal transducer and activator of transcription (STAT3), while imidazolium oximes had no effect. The oxime VII also did not change the phosphorylation of cellular energy sensor AMP-activated protein kinase (AMPK) or its downstream substrate acetyl-CoA carboxylase (ACC). In contrast, K867 markedly decreased the phosphorylation of AMPK and increased the phosphorylation of ACC. The oxime X increased the phosphorylation of both AMPK and ACC, which points to the activation of the AMPK-signalling pathway.

Figure 3

In order to find whether there is a predicted target class for our tested oximes in the existing database, we ran them through the SwissTargetPrediction web tool (23) based on structural similarities with molecules of known activities. Results presented in Figure 4 indicate high probability of interaction between our oximes and the members of the G-coupled membrane receptors (GPCR) family. Some oximes are also likely to interact with several other targets, such as specific enzymes, nuclear receptors, or proteases.

Figure 4

Figure 5 shows the potential of our oximes to interact with acetylcholine receptors (AChRs). Compared to control, all but K870 nearly completely suppressed the activity of ACh and the increase in iCa²⁺caused by the activation of muscarinic G-protein-coupled receptors and nicotinic ionotropic channels.

Figure 5