Selenotriapine – An isostere of the most studied thiosemicarbazone with pronounced pro-apoptotic activity, low toxicity and ability to challenge phenotype reprogramming of 3-D mammary adenocarcinoma tumors

2.1 Reagents and instrumentation

tert-Butyl (2-formylpyridin-3-yl)carbamate and 3-AP were synthesized according reported procedures (Kowol et al., 2009). All other chemicals and solvents (reagent grade) were obtained from commercial suppliers and used without further purification. Elemental analyses (C, H, N) were performed by the standard micromethods using the ELEMENTAR Vario EL III CHNS/O analyzer at Center for Instrumental Analysis, Faculty of Chemistry – University of Belgrade. Elemental analyses are within ±0.4%, confirming >95% purity. IR spectra were recorded on a Thermo Scientific Nicolet 6700 FT-IR spectrophotometer by the Attenuated Total Reflection (ATR) technique in the region 4000–350 cm^–1. Abbreviations used for IR spectra: vs very strong; s, strong; m, medium; w, weak. The NMR spectra were performed on a Bruker Avance 500 equipped with broad-band direct probe. Chemical shifts are given on δ scale relative to tetramethylsilane (TMS) as internal standard for ¹H and ¹³C, or indirect referencing to TMS as ¹H standard for ⁷⁷Se. All spectra were measured at 298 K. Abbreviations used for NMR data: s, singlet; m, multiplet; py, pyridine; Cq, py = pyridine quaternary carbon atom. pH values were measured using a CRISON pH-Burette 242S equipped with a CRISON 50 29 micro-combined pH electrode (CRISON INSTRUMENTS, S.A. Spain). For acidity constants determination, UV/vis spectra were recorded on a Thermo Scientific Evolution 60S spectrophotometer (Thermo Fisher Scientific Inc, Waltham, Massachusetts, USA) using a quartz cell with 1.0 cm path length. For DPPH scavenging activity, absorbance at 517 nm was measured using a Thermo Scientific Appliskan. Anticancer related experiments were performed on Guava® easyCyte 12HT Benchtop flow microcapillary cytometer (EMD Millipore, Darmstadt, Germany) using the InCyte® software package (EMD Millipore, Cat. No. 0500-4120). A Submarine Mini-gel Electrophoresis Unit (Hoeffer HE 33) with an EPS 300 power supply was used for DNA cleavage studies. The stained gel was illuminated under a UV trans illuminator Vilber-Lourmat (France) at 312 nm and photographed with a Nikon Coolpix P340 Digital Camera through filter Deep Yellow 15 (Tiffen, USA). For HSA binding experiments UV/vis spectra were recorded on a UV-1800 Shimadzu spectrophotometer (Shimadzu, Japan) equipped with thermostat bath, against the corresponding blank (0.1 M PBS pH 7.4) in the range 250–450 nm. The fluorescence measurements were performed on a FluoroMax-4 Jobin Yvon (Horiba Scientific, Japan) spectrofluorometer equipped with 1.0 cm quartz cell and thermostat bath. The excitation and emission slit widths were set to 6.5 nm.

2.2 Synthesis of 3-aminopyridine-2-carbaldehyde selenosemicarbazone (Se-3-AP)

To a mixture of tert-butyl (2-formylpyridin-3-yl)carbamate (1.468 g, 6.61 mmol) and selenosemicarbazide (662 mg, 7.27 mmol) in EtOH/H₂O solvent mixture (22.5 mL, 67% v/v), 3 mL of conc. HCI was added. The resulting solution was refluxed for 3 h, cooled down to a room temperature (RT) and resulting precipitate was filtered of. The crude yellowish Se-3-AP × HCl was transferred into a flask and 40 mL of hot water and 10% aq. NaHCO₃ (8 mL) was added. The mixture was stirred at RT for 1 h (at pH 7.0). The crude product was filtered of and washed with water (10 mL), EtOH (3 mL) and Et₂O (10 mL). The obtained solid was dried under high vacuum for a few hours. Yield: 0.28 g (66%). Anal. Calcd. for C₇H₉N₅Se (M_r 242.14): C, 34.72; H, 3.75; N, 28.92. Found: C, 34.52; H, 3.61; N, 28.90%. UV/vis (DMSO), λ_max, nm (ɛ, M⁻¹ cm⁻¹): 316 (5947), 393 (21248). IR (ATR, cm^–1): 3398 (vs), 3300 (vs), 1626 (vs), 1542 (s), 1451 (s), 1366 (m), 1318 (w), 1266 (s), 1147 (m), 1053 (w), 803 (w), 749 (w). ¹H NMR (500 MHz, DMSO‑d₆): δ 11.57 (s, 1H, N—NH), 8.58 (s, 1H, Se⚌C—NH₂), 8.45 (s, 1H, Se⚌C—NH₂), 8.43 (s, 1H, HC⚌N), 7.84 (s, 1H, H_py), 7.19–7.04 (m, 2H, H_py), 6.47 (s, 2H, NH₂). ¹³C NMR (126 MHz, DMSO‑d₆): δ 172.33 (C⚌Se), 150.61 (C⚌N), 144.17 (C_q, C_pyCH), 137.28(C_py), 132.45 (C_q, C_pyNH₂), 124.69 (C_py), 122.37 (C_py). ⁷⁷Se NMR (95 MHz, DMSO‑d₆): δ 203.50.

2.3 Cyclic voltammetry

The cell (10 mL) consisted of three-electrode system: glassy carbon electrode (inner diameter of 3 mm; CHI 104), an Ag/AgCl (saturated KCl) reference electrode and Pt counter electrode. The potential was swept over the range from 2.0 to +1.2 V (vs. Ag/AgCl) at scan rate of 100 mV s⁻¹. Measurements were performed at RT with deaeration by passing a stream of nitrogen through the solution for 5 min prior to the measurement and then maintaining a blanket atmosphere of nitrogen over the solution during the measurements. The potentials were measured in 0.10 M tetrabutylammonium hexafluorophosphate/DMSO, and are quoted relative to Ag/AgCl reference electrode.

2.4 Determination of acidity constants

Acidity constants of Se-3-AP were determined by spectrophotometric titration at t = 25 ± 1 °C. Working solution (c = 0.031 mM) was prepared by dissolving accurately weighted dried Se-3-AP in 5 mL of DMSO; 10 mM phosphate buffer (pH 1.77, I = 0.1 M NaCl) was added up to 100 mL. First, pH value was adjusted to 1.65 with conc. HCl, than small volume increments of 1 M KOH were added stepwise, until pH 11.88 was reached (total volume change at the end of the titration was lower than 2%). During titration, after each pH value equilibration, pH values were continuously measured and UV/vis spectra were recorded. The spectra were recorded at following pH values: 1.73, 1.98, 2.25, 2.50, 2.82, 3.19, 3.70, 3.89, 4.09, 4.33, 4.55, 4.88, 5.21, 5.50, 5.82, 6.49, 9.19, 9.58, 9.79, 9.98, 10.20, 10.50, 10.95, 11.41, 11.88, against the phosphate buffer as a blank. The pH electrode was calibrated by standard CRISON buffer solutions (pH 4.01, 7.00, and 9.21). Acidity constants were calculated according to transformed forms of classical spectrophotometric equations (Albert and Serjeant, 1984).

2.5 Free radical scavenging antioxidant assay

The proton donating ability of Se-3-AP and 3-AP was assayed using a protocol for the determination of radical scavenging activity (Prior et al., 2005). Compounds were dissolved in pure DMSO and were diluted into ten different concentrations. Commercially available free radical DPPH was dissolved in methanol at a concentration of 6.58 × 10^–5 M. Into a 96-well microplate, 140 µL of DPPH solution was loaded and 10 mL DMSO solution of the tested compounds was added, or pure DMSO (10 µL) as the control. The microplate was incubated for 30 min at 298 K in the dark and the absorbance was measured at 517 nm. All the measurements were carried out in triplicate. The scavenging activity of the compounds was calculated using Eq. (1):

IC₅₀ values were calculated from the plotted graph of scavenging activity against the concentrations of the samples. IC₅₀ is defined as the total antioxidant concentration necessary to decrease the amount of the initial DPPH radical by 50%. IC₅₀ was calculated for all compounds based on the percentage of DPPH radicals scavenged. Vitamin C was used as the reference compound (positive control) with concentrations 50–500 μg × mL^–1.

2.6 Cell cultures

Human mammary adenocarcinoma (MCF-7, ATCC® HTB-22) cell line was maintained in Dulbecco's Modified Eagle's high glucose Medium (DMEM, Dominique Dutscher, 67172 Brumath cedex, France, Cat No L0102-500), while human acute monocytic leukemia cell line (THP-1, ATCC® TIB-202) was maintained in Roswell Park Memorial Institute 1640 medium (RPMI-1640, Life Technologies, Paisley, UK, Cat. No. 11875-093), supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS, Life Technologies, Paisley, UK, Cat No 10270-106) and 1% (v/v) penicillin-streptomycin (10,000 U/mL and 10,000 µg/mL Life Technologies, Paisley, UK, Cat No 15140-122). Cells were kept at 37 °C in humidified atmosphere containing 5% (v/v) CO₂ during their exponential growing phase and in the course of incubation with investigated compounds. Investigated compound was initially dissolved in DMSO to the stock concentration of 20 mM. Further dilutions to the experimental concentrations applied on the cells have been done with RPMI-1640 or DMEM media immediately before each experiment, thus the final concentration of DMSO on cells treated with the highest applied concentration was 0.5% (v/v).

2.7 Evaluation of pro-apoptotic activity

Cells were seeded in 96 flat bottom well plates (Corning® Costar®, Cat. No. CLS3596) in a volume 0.1 mL, at a density of 10,000 per well. MCF-7 cells were left overnight to settle, while treatment of THP-1 cells started 2 h after seeding. Investigated compounds were added in a range of six concentrations. As controls, non-treated cells, cells treated with 0.5% DMSO, and cells treated with Celastrol (Enzo Life Sciences, Cat. No. ALX-350-332-M025) at 50 µM concentration were used. After 24 h of incubation, from wells with treated MCF-7 cells supernatant medium with non-adherent cells were removed into another 96 well plate. Fresh PBS was added to remaining adherent cells afterwards the plate was centrifuged at 450g for 10 min. Supernatant was discarded and 200 µL of trypsin-EDTA (BioWest, Nuaille, France, Cat No L0930-100) was added to each well. Cells were detached in about 15 min of incubation at 37 °C. Trypsin-EDTA was removed after additional spinning cycle, afterwards previously removed supernatants with non-adherent cells were added to trypsinized cells and stained with Annexin V-FITC (Immuno Tools, Friesoythe, Germany, Cat No 31490013) and PI (Miltenyl Biotec Inc, Auburn, USA, Cat No 130-093-233) in volumes of 3 µL. In the case of THP-1 cells, Annexin V-FITC and PI were added to the wells with cells right after the incubation time was over. Described trypsinization protocol was applied each time MCF-7 cells were prepared for flow cytometry analyses, unless is stated otherwise. Plates were analyzed and classified according to AnnexinV-FITC (green fluorescence) and PI (red fluorescence) labeling on viable (double negative), pre-apoptotic cells (Annexin V-FITC single-stained cells), necrotic cells (PI single-stained cells), and cells in advanced phases of apoptosis (double-stained cells).

2.8 ED₅₀ determination

Percentages of Annexin V single-stained and double-stained cells were summarized for each concentration of investigated compound. The computed percentages were potted against corresponding concentrations on a concentration-response graph. ED₅₀ concentration was calculated as the one that corresponds to a half-way of the sigmoidal concentration-dependent curve using asymmetric five-parameter logistic equation (GraphPad Prism 6 software).

2.9 Cell cycle analysis

Distribution of cells within phases of mitotic division has been evaluated on remaining cells after Annexin V-FITC/PI analysis. Right after pro-apoptotic read out was finished, cells were fixed in EtOH overnight at 4 °C, and afterwards stained with FxCycle PI/RNAse Staining solution (Molecular Probes, Cat. No. F10797), and analyzed on a microcapillary flow cytometer.

2.10 Inhibition of caspase activity

Cells were treated with investigated compound at ED₅₀ concentration for 6 h with or without pan-caspase inhibitor Z-VAD-fmk (Promega, Madison, USA, Cat. No. G7232). As controls, non-treated cells, cells treated with Z-VAD-fmk only, and cells treated with ED₅₀ concentration only were used. After incubation period was ended, treated cells were carried out for Annexin V/PI staining as described above, and analyzed on a cytometer. The percent of apoptosis inhibited by Z-VAD-fmk co-treatment was determined by Eq. (2):

2.11 Evaluation of caspase-8 and -9 activities

Cells were treated with investigated compound at ED₅₀ concentration for 6 h afterwards activity of caspase-8 and -9 were assayed by means of Guava Caspase 9 SR and Caspase 8 FAM kit (EMD Millipore, Cat. No. 4500-0640), following manufacturer’s instructions. Cells were analyzed on flow microcapillary cytometer. In acquired data cells were discriminated according to expression of activated caspase-8, caspase-9, or both.

2.12 Assessment of changes in mitochondrial transmembrane potential

Cells were treated over 6 h with investigated compound in concentration of 50 µM. After incubation was terminated, cells were trypsinized and stained with FlowCellect MitoDamage Kit (Merck Millipore Corporation, Darmstadt, Germany, Cat. No. FCCH100106) according to manufacturer’s recommendations. Analysis has been performed on flow microcapillary cytometer. Cells were classified according to labeling with Annexin V-FITC, MitoSense Red, and 7-AAD.

2.13 Determination of mitochondrial superoxide radical generation

Cells were treated over 6 h with investigated compounds in concentration of 50 µM, afterwards were stained with MitoSox Red (Molecular Probes, Cat. No. M36008) according to manufacturer’s recommendations. Analysis has been performed on a flow microcapillary cytometer.

2.14 Growth inhibition of 3-D tumor models

3-D MCF-7 tumor models were made in 96 well plates (Corning, Sigma-Aldrich, St. Louis, Mo, USA, Cat No 4515). Tumors were left to grow for additional four days, afterwards investigated compound was added in concentrations of 1, 10, and 100 µM. Evaluation has been maintained during 8-day incubation period, with media exchanged on day 4. Changes in the tumors sizes have been assessed on Celigo® imaging cytometer estimating the area of spheroids’ cross section (Cyntellect, Brooks Life Science Systems, Poway, CA, USA) using Celigo software. Growth rates of non-treated and treated spheroids were computed for each day during 8-day incubation by dividing the area on the day n with the area on the day 0.

2.15 Detection of gene expression in live cells

After 8-day incubation, non-treated and treated spheroids were transferred to flat bottom 96 well plate (Corning® Costar®, Cat. No. CLS3596), and segregated by trypsin in several consecutive courses (BioWest, Nuaille, France, Cat No L0930-100) with gently repeated pipetting. After spheroids were completely dissociated into single cell suspensions, samples were treated according to recommendations of manufacturer with SmartFlare RNA probes (Millipore, Merck, Darmstadt, Germany) or not (non-stained control): scrambled control (negative control, Cat No SF-102), uptake control (Cat No SF-114) and Oct-4 (Cat No SF-460). Incubation with SmartFlare probes lasted for 16 h at 37 °C in humidified atmosphere containing 5% (v/v) CO₂, afterwards all samples were trypsinized and analyzed on a flow microcapillary cytometer.

2.16 DNA damage experiments

The ability of 3-AP and Se-3-AP to inhibit Cu(I)-driven plasmid DNA damage was assayed using a literature protocol (Battin et al., 2011). Breafly, CuSO₄ (6 μM), the indicated concentrations of 3-AP/Se-3-AP, EtOH (10 mM), deionized H₂O, 3-(N-morpholino)propanesulfonic acid (10 mM), NaCl (130 mM), and ascorbic acid (1.25 equiv; 7.5 mM) were mixed at pH 7 and allowed to stand for 5 min at room temperature. Plasmid (pUC19, 0.1 pmol in 10 mM TrisHCl pH 8) was added to each reaction mixture and the solutions were again allowed to stand for additional 5 min. The hydroxyl radical formation was initiated by the addition of H₂O₂ (50 mM). After 30 min, EDTA (50 mM) was added to quench the reactions.

2.17 Determination of metHb

Blood sample from healthy human donor was collected in Vacutainer collection tubes (BD, Plymouth, UK) and used immediately. RBC were isolated by centrifugation (480g for 5 min at 4 °C) and then were washed in physiological solution. To prepare RBC lysates, RBC were lysed with miliQ water, the debris was removed by centrifugation (13000g for 30 min at 4 °C), and the supernatant was used ([oxyHb] = 4.4 mM). Working solutions of oxyHb (1.5 and 0.1 mM) were prepared by dilution using 0.1 mM PBS (pH 7.4). Stock solutions of Se-3-AP (5 mM) and 3-AP (5mM) were prepared by dissolving an appropriate amount of substance in DMSO and further diluted in 0.1 mM PBS (pH 7.4). The total content of DMSO in tested samples was less than 0.5% (vol.%). Concentrations of oxyHb and metHb were determined at 577 and 630 nm.

2.18 UV/vis and fluorescence spectroscopy

The working solutions of Se-3-AP different concentrations, as indicated in the text, for UV/vis and fluorescence spectroscopy were prepared by diluting the stock solution of Se-3-AP (5 mM) with 0.1 M PBS (pH 7.4) or DMSO. A stock solution of Se-3-AP (5 mM) was prepared daily by dissolving an appropriate amount of substance in DMSO.

2.19 HSA binding experiments

Fatty acid free HSA (<0.007% fatty acids, M_w 66 478 Da) was purchased from Sigma. A stock solution of HSA (0.25 mM) was prepared by dissolving an accurately weighed mass of HSA in freshly prepared 0.1 M PBS (pH 7.4), and kept in a freezer in 100 µL portions. A stock solution of Se-3-AP (5 mM) was prepared daily by dissolving an appropriate amount of substance in DMSO. Millipore water was used for all aqueous solutions.

Binding of Se-3-AP to HSA was studied by fluorescence quenching titration method using the intrinsic fluorescence of HSA as probe. For fluorescence studies, solutions of HSA and Se-3-AP were prepared daily by diluting the stock solutions of HSA and Se-3-AP with 100 mM PBS pH 7.4 or DMSO to the final concentrations of HSA (0.125 µΜ) and Se-3-AP (80 µM), respectively. Before recording the fluorescence spectrum, diluted HSA solution was ultrafiltered using filters with 0.45 μm pore size. Small aliquots of 80 μM Se-3-AP solutions were added to 2.5 mL of 0.125 μM HSA solution. The Se-3-AP was added in 1.5, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 M equivalents, so after the last equivalent, the total DMSO did not exceed 2.4% in volume. After each aliquot addition, the system was stirred and left to equilibrate for 5 min before fluorescence emission spectra were recorded in the range 300–450 nm with an excitation wavelength of 280 nm and 0.1 s integration time. Spectra represent the average of three accumulations and the background buffer signal was subtracted from each spectrum. Fluorescence quenching data were corrected for the inner-filter effect and further processed (for details see Supplementary data).

2.20 Docking to HSA

Se-3-AP was optimized at B97D DFT level of theory (Grimme, 2006), using LanL2DZ basis set for Se (Dunning and Hay, 1977; Hay and Wadt, 1985), and 6-311G+(d,p) basis set for C, N and H atoms (Krishnan et al., 1980; McLean and Chandler, 1980). Geometrical optimization was carried in Gaussian 09, revision D.01 (Frisch et al., 2016). Ligand was afterwards prepared for docking in ADT Tools 1.5.6 (Morris et al., 2009; Sanner, 1999). Three HSA crystal structures (PDB IDs: 1BJ5 (Curry et al., 1998), 2BXD (Ghuman et al., 2005), and 4L9Q (Wang et al., 2013) were obtained from the Protein Data Bank (http://www.rcsb.org/pdb). The active sites were identified according to positions of crystallized ligands in the PDB structures. Ligands were removed, as well as water and ions and structures were prepared for docking using Autodock Tools 1.5.6 (Morris et al., 2009; Sanner, 1999). Docking was carried in AutodockVina 1.1.2 (Trott and Olson, 2010). The grid box size was set to 24 × 24 × 24 Å and exhaustiveness to 250. All calculations were carried on PARADOX computer cluster (Scientific Computing Laboratory of the Institute of Physics, Belgrade, Serbia).

2.21 Artemia salina toxicity assay

A teaspoon of lyophilized eggs of the brine shrimp Artemia salina was added to a vessel filled with 1 L of the artificial sea water containing several drops of yeast suspension (3 mg of dry yeast in 5 mL of distilled water). The vessel was partially exposed to illumination and with assisted water aeration kept for 48 h at 28 °C. After hatching, nauplii were collected by pipette at the illuminated side of the vessel. Tested compounds were dissolved in DMSO (1 mg/100 µL) immediately before the experiment. Five working concentrations have been made with artificial seawater and added to vials which contained 10–20 hatched nauplii, thus the final concentrations of investigated compounds were in the range 0.01–5 mg/mL. K₂Cr₂O₇ in the same concentration range served as positive control. Vials with nauplii, sea water and DMSO in concentrations that corresponded to those in experimental samples (0.1–5%) served as a negative control. All vials were left at 28 °C, under illumination, and water aeration for 24 h, afterwards live and dead nauplii were counted. The LC₅₀ concentration was determined as the one that induced death of 50% nauplii in treated population. Each experimental sample has been performed in duplicates.

2.22 Statistical analysis

Analyzed data were expressed in three replicates each obtained from different experiments. The statistical significance of differences was assessed using unpaired t test with Welch’s correction, or one-way ANOVA followed by unpaired t test with Welch’s correction depending on number of groups to compare, which is specifically notified in figure captions.

3.1 Chemistry

The three step synthesis of Se-3-AP, with an overall yield of 66%, started from 3-amino-2-bromopyridine (Scheme 1). The protection of amino group with ditert-butyl dicarbonate (Boc₂O) in dry THF was achieved using sodium bis-(trimethylsilyl)amide as a base (Kowol et al., 2009). The tert-Boc protected 3-amino-2-bromopyridine was than treated with n-butyllithium (n-BuLi) in dry THF to obtain lithiated species, which was further converted to carboxaldehyde by treatment with N-formylpiperidine (Kowol et al., 2009). Condensation reaction between ethanolic solutions of tert-Boc protected 3-amino-2-formylpyridine carboxaldehyde and selenosemicarbazide in the presence of conc. HCl afforded Se-3-AP × HCl. Hydrochloride salt was converted into Se-3-AP by treatment with sodium bicarbonate. 3-AP was synthesized as previously reported (Kowol et al., 2009).

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Scheme 1

Three step synthesis of Se-3-AP. Reagents and conditions: (i) sodium bis(trimethylsilyl)amide (1 M, THF), (Boc)₂O; (ii) n-BuLi, N-formylpiperidine; (iii) selenosemicarbazide, conc. HCl, NaHCO₃.

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Characterization of Se-3-AP was done by means of UV/vis, fluorescence and 1-D and 2-D NMR spectroscopy (Figs. S1–S8, Supplementary data). In ¹H NMR spectrum of Se-3-AP in dimethylsulfoxide (DMSO)-d₆ just one set of signals could be observed, with the N-NH signal at 11.57 ppm. This indicates, as previously observed for 3-AP and related 2-formylpyridine thiosemicarbazones (Kowol et al., 2016; Pessoa et al., 2001), that Se-3-AP exists in E-isomeric form. In the UV/vis spectrum of Se-3-AP absorptions at 316 nm (weak) and at 393 nm (strong) were found, while there is a maximum at 457 nm in emission spectrum irradiated at λ_ex = 360 nm in DMSO. The redox behavior of Se-3-AP and 3-AP has been investigated by cyclic voltammetry in the potential range from +1.2 to –2.0 V relative to Ag/AgCl reference electrode. In the cyclic voltammogram of both Se-3-AP and 3-AP (Fig. S9, Supplementary data) there is one well defined oxidation peak (Se-3-AP: +0.661 V; 3-AP: +0.628 V) and one reduction peak (Se-3-AP: –0.857 V; 3-AP: –0.951 V).

The proton-dissociation processes of Se-3-AP were followed by UV/vis spectrophotometric titration. Since Se-3-AP is poorly soluble in water, 5% (v/v) DMSO/water solvent mixture was found to be suitable media for aqueous solution equilibrium study. UV/vis spectra of Se-3-AP in the pH ranges 1.73–6.49 and 6.49–11.88 are shown in Fig. 1. Two proton-dissociation processes could be determined (Scheme 2) with two well separated (ΔpK_a > 3) pK_avalues (pK_a1 = 4.23 ± 0.01 at 404 nm and pK_a2 = 9.75 ± 0.04 at 296 nm), as indicated by clearly visible isosbestic points in the two pH regions. Characteristic spectral changes of Se-3-AP can be observed in the 250–450 nm wavelength range. Se-3-AP displays two intense absorption bands due to n → π^∗ transitions of the pyridine ring and the selenosemicarbazide moiety (Filipovic et al., 2016). During the first deprotonation step ([H₂A]⁺ → [HA]), a blue shift and a decrease in intensity of the absorption maximum in the visible region and a redshift and a decrease in absorption maximum in the UV region of the spectra were observed (Fig. 1A). The second deprotonation step ([HA] → [A]^–) was accompanied by a small redshift and slight decrease in intensity of the absorption maximum in the visible region and a decrease in absorption maximum without changes in its position in the UV region of the spectra (Fig. 1B). As can be seen from concentration distribution curves (Fig. 1C) the neutral form of Se-3-AP predominates in the physiological pH range. It should be noted that the determined constants represent macro ionization constants. As shown in Scheme 1, K_a1 represents the dissociation of the protonated pyridinium unit, while K_a2coresponds to deprotonation of the hydrazone nitrogen atom of the selenosemicarbazide moiety. K_a2 accounts for different contributions of the selenone tautomer (shown in Scheme 2) and selenenol tautomer (not shown).

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Fig. 1

UV/Vis absorption spectra of Se-3-AP [c = 0.031 mM; t = 25.0 °C, I = 0.10 M (NaCl) in 5% (v/v) DMSO/H2O] recorded in the pH ranges 1.736.49 (A) and 6.4911.88 (B). Spectra of pure H2A+, HA and A– forms and isosbestic points are indicated. Se-3-AP distribution diagram (C).

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Scheme 2

Proteolytic equilibria of Se-3-AP in aqueous media. HA, as neutral form of Se-3-AP, is represented in one tautomeric form, while all tautomers coexist in solution.

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The proton donating ability of Se-3-AP and 3-AP was assayed using a protocol for the determination of radical scavenging activity, the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method (Prior et al., 2005). IC₅₀ values were calculated from the plotted graph of scavenging activity against the concentrations of the samples. IC₅₀ is defined as the total antioxidant necessary to decrease the initial DPPH radical concentration by 50%. Vitamin C was used as the reference compound (positive control). The obtained IC₅₀ values (in mM) are as follows: 1.13 ± 0.01, 3-AP; 0.107 ± 0.001, Se-3-AP; 0.079 ± 0.002, vitamin C. These results showed that Se-3-AP is one order of magnitude more potent antioxidant than 3-AP.

3.2 Anticancer activities

3-AP and Se-3-AP have been evaluated in terms of their ability to induce cell apoptosis in THP-1 and MCF-7 malignant cell lines. Compounds were added in a range of six concentrations and left on cells for 24 h, afterwards percentages of live and dead cells were determined by means of Annexin V/propidium iodide (PI) dual staining assay. Both, 3-AP and Se-3-AP have proven to be strong inducers of cell death in THP-1 cells but with apparently different activity. Treatment of THP-1 cells with 3-AP administered in a span from 1–100 µM predominantly induced accumulation of double stained events in the quite high percentage already at the lowest employed concentration, with a concentration-dependent increase throughout the concentration range (Fig. 2A, left panel). At the same time, percentages of Annexin V single-stained cells that represent events in the early phase of apoptotic death, has remained at the level of untreated control, while incidence of necrotic cells remained above 10% and in almost all treated samples. Since Annexin V/PI double stained cells can be interpreted as cells in advanced phases of apoptosis or necrosis, the coincidental lack of early apoptotic events clearly indicates that treatment with 3-AP induced a vigorous necrotic response in THP-1 cell line. For that reason 3-AP is considered to possess an unfavorable mode of activity and was not included in further evaluations on this cell line. On the other hand, treatment of THP-1 cells with Se-3-AP in initial range from 1–100 µM also induced a major accumulation of double-stained cells, but those were accompanied by significant percentages of cells at the initial phase of apoptotic death (data not shown). Considering the incidences of cell death were too high in the span of applied concentrations describing only the top plateau of the concentration–response sigmoidal curve so that ED₅₀ concentration could not be determined, it was necessary to decrease concentration range of Se-3-AP on THP-1 cells half the way down to 0.5–30 µM. As represented in Fig. 2B, treatment with Se-3-AP gradually and concentration-dependently increased percentages of cells in early and late phases of apoptosis, whereas incidence of necrosis was preserved on the level of untreated control. Those data described sigmoidal concentration-response curve with a wide slope while the top plateau begins at the concentration of 30 µM (Fig. S10A, Supplementary data). Such a curve is characteristic of a drug which dose can be accurately titrated to achieve a desired outcome while easily avoiding any toxic effect.

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Fig. 2

(A) Types of cell death in THP-1 (left panel) and MCF-7 cells (right panel) treated with 3-AP. Results are represented as percentages of stained cells from a single experiment considering the replicates have not been acquired due to high toxicity or lack of activity. (B) Types of cell death (left panel) and changes in cell cycle distribution (right panel) in THP-1 cells treated with Se-3-AP. (C) Types of cell death (left panel) and changes in cell cycle distribution (right panel) in MCF-7 cells treated with Se-3-AP. Types of cell death in each experiment were determined after 24 h incubation by Annexin V/PI double staining. Changes in cell cycle distribution on both cell lines have been assessed on the same cell samples used for Annexin V/PI read out. Incidences of cells found in G0/G1, S and G2/M phases were determined according to non-treated control population. Results in (B) and (C) are expressed as the mean ± SD (n = 2 independent replicates).

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Treatment of MCF-7 cells with 3-AP (1–100 µM) resulted in poor frequency of cell death while the majority of those events were also in the process of necrotic death (Fig. 2A, right panel). On the contrary, Se-3-AP on the same cell line induced strong apoptotic response with concentration–response curve nicely defined within applied range of 1–100 µM (Fig. 2C and Fig. S10B Supplementary data). However, concentration–response curve, which describes activity of Se-3-AP on MCF-7 cell line, distinguishes from that defined for THP-1 cells by the slope positioned within a narrowed concentration span, while the top plateau started already from the concentration of 50 µM (Fig. S10A, Supplementary data).

Changes of cell distribution within phases of mitotic division in THP-1 cells caused by the treatment with Se-3-AP have shown moderate concentration-dependent variations (Fig. 2B, right panel). Moderate accumulation of dividing cells at the G1-to-S phase of the cell cycle was evident up to the concentration of 3 µM, when a gradual decrease in percentage of cells at the S phase was recorded. Thus in the samples treated with Se-3-AP at 30 µM incidence of cells at the S phase returned to the level of non-treated control accompanied with discrete accumulation of cells at the G2/M phase. Gathering of cells at the S phase was also found in the MCF-7 treated samples (Fig. 2C, right panel). However, Se-3-AP at concentration of 1 µM aroused formation of S-to-G2 arrest that already at the next concentration level of 10 µM shifted into cell collecting at the G1-to-S phase. Considering necrosis was the dominant form of cell death in THP-1 cells treated with 3-AP, changes in cell cycle progression were not assessed due to their irrelevance.

Although 3-AP showed weak pro-apoptotic activity in MCF-7 cells, so that what type of changes it induced at 10, 50 and 100 µM only were evaluated (Fig. S11, Supplementary data). Incubation of MCF-7 cells with 3-AP resulted in a slight accumulation of cells at the G2/M phase instead at the G1-to-S that was seen after treatment with Se-3-AP (Fig. 2B and C).

In order to evaluate the role of caspase activation in apoptotic death of THP-1 and MCF-7 cells induced by the treatment with Se-3-AP, cells were incubated with this compound at the ED₅₀ concentration with and without the presence of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk). After 6 h of treatment, cells were analyzed by means of Annexin V/PI dual staining method, and changes in incidences of apoptotic and necrotic events between samples treated with Se-3-AP alone and co-incubated with Z-VAD-fmk were computed. As represented in Fig. 3A, the addition of pan-caspase inhibitor almost completely inhibited apoptosis induced by Se-3-AP in both cell lines. Nevertheless, the lack of caspase activity induced treated cells to necrosis. This result demonstrates that apoptosis in THP-1 and MCF-7 cells was highly dependent upon caspase activation. The only dissimilarity observed between those two cell lines concerns different caspase-dependency of apoptotic death initiation, which may indicate on possibility that Se-3-AP in MCF-7 cell also can trigger caspase-independent apoptosis.

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Fig. 3

(A) Impact of pan-caspase inhibitor Z-VAD-fmk on incidences of apoptotic and necrotic cell deaths in THP-1 and MCF-7 cells treated with Se-3-AP applied at ED50 concentration. (B) Percentage of THP-1 and MCF-7 cells with activated caspase-8 or/and caspase-9 due to treatment with Se-3-AP applied at ED50 concentration. All results are presented as the mean ± SD (n = 3 independent replicates).

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Further, we wanted to determine which caspase pathway was primarily activated in THP-1 and MCF-7 cells by the treatment with Se-3-AP. For that reason, cells were treated with Se-3-AP at its ED₅₀ concentration and assayed after 6 h incubation for caspase-8 and -9 activities. Results are presented as average percentage of cells that express activated either caspase-8 or caspase-9 alone, and an average percentage of cells that express both activated caspases (Fig. 3B). In non-treated THP-1 samples, a basic level of activated caspase-9 exits (5.9 ± 0.3% of cells), while percentages of cells with activated caspase-8 only or both caspases were very low (0.5 ± 0.4% and 0.4 ± 0.3%, respectively). Quite the opposite, percentage of non-treated MCF-7 cells with primarily activated caspase-8 (3.3 ± 0.6%) and both activated caspases (3 ± 1%) dominated over activation of caspase-9 (0.6 ± 0.1%).

For Se-3-AP treated THP-1 samples high but non-significant increase in percentage of cells positive for activated caspase-9 only was found (20 ± 4%), whereas a high level of significance was determined comparing cells which displayed activated form of both caspases. Results of this assay definitely reveal that the dominant mechanism of apoptosis triggered by Se-3-AP in THP-1 cells involves activation of intrinsic apoptotic pathway, whereas activation of caspase-8 arises as a result of cross-talk between these two apoptotic pathways. Activation of caspase-9 was the mainstream event in MCF-7 cells too. However, here the cross-talk between intrinsic and extrinsic apoptotic pathways evidently did not play any important role in signal transduction during apoptotic process.

Capacity of Se-3-AP to induce generation of mitochondrial superoxide radicals (O₂^–•) has been evaluated on both cell lines. Percentages of THP-1 and MCF-7 cells positive for mitochondrial O₂^–• have been evaluated by MitoSox Red in cells after 6 h incubation with Se-3-AP [50 µM]. Results were analyzed with regard to following parameters: percentage of O₂^–•-positive cells, and median fluorescence intensity (MFI) determined for O₂^–•-positive subpopulation of cells expressed in arbitrary units (AU). MFI is relevant of the quantity of generated O₂^–• per cell. Percentages of O₂^–•-positive cells in non-treated controls, THP-1 and MCF-7 cell lines are different in their basal potential for generating mitochondrial O₂^–• (5.2 ± 0.7 AU and 20 ± 7 AU for THP-1 and MCF-7 cells, respectively), while MFI values for those controls were almost the same (Fig. 4A and B). After addition of Se-3-AP to THP-1 cells, percentage of O₂^–• positive cells increased three fold, but the median amount of generated O₂^–• per cell remained at the level of non-treated control. On the contrary, treatment of MCF-7 cells with Se-3-AP doubled the percentage of O₂^–•-generating cells, whereas the amplitude of superoxide production per cell was significantly higher than in control population. This result demonstrates the phenotype-specific response to Se-3-AP regarding mitochondrialO₂^–• generation.

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Fig. 4

(A) Percentages of THP-1 and MCF-7 cells positive for mitochondrial O₂^–• after 6 h treatment with Se-3-AP [50 µM], as determined by MitoSox Red. Results are presented as the mean ± SD (n = 3 independent replicates). (B) MFI of MitoSox Red-stained cells giving a median quantity of produced O₂^–• per cell. Statistical significance has been determined by unpaired t test with Welch’s correction (n = 3 independent experiments). (C) Changes of mitochondrial membrane potential in MCF-7 cells after 6 h treatment with Se-3-AP [50 µM], as determined by MitoSense Red. Results are presented as the mean ± SD percentage of cells discriminated according to labeling by MitoSense Red, Annexin V and 7-AAD (n = 2 independent replicates).

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Following the insight that treatment with Se-3-AP significantly increased mitochondrial O₂^–• production in MCF-7 cells only, together with the fact that activation of intrinsic apoptotic pathway was the executive in the process of their apoptotic death, it was decided to evaluate the loss of mitochondrial transmembrane potential (MTP) in this cell line. Evaluation of MTP dissipation has been assessed after 6 h incubation of MCF-7 cells with Se-3-AP (c = 50 µM). Chosen concentration being the same as for determination of mitochondrial O₂^–• production, allowing an unbiased estimation whether generation of O₂^–• was the main cause for activation of intrinsic apoptotic pathway. As seen in Fig. 4C, almost all the cells in non-treated controls with altered MTP were additionally single-stained with 7-AAD indicating necrotic death. Compared to control, treatment with Se-3-AP induced about a twofold increase in percentage of cells with dissipated MTP, whereas regarding their staining with Annexin V and 7-AAD a similar distribution was recorded as in non-treated control. Majority of Se-3-AP-treated cells that showed loss of MTP were 7-AAD single-stained, while only few of them were labeled with Annexin V alone. Considering the short incubation time and significantly small percentage of cells found in early apoptosis (Annexin V-single stained events), necrosis remains the main way leading to cell death.

The treatment impact on MCF-7 3-D spheroids was monitored in regard of changes in size and morphology. Insufficient mass transport of oxygen, nutrients and metabolites through cellular barriers of spheroidal structure clearly distinguishes three concentric zones: necrotic core (dark sphere located in the center of 3-D culture consisting of dead cells), quiescent zone (transparent dark gray rim that surrounds necrotic core, consisting of living non-dividing cells) and the proliferating zone (transparent border located on the outer edge). For non-treated spheroids, sizes and mutual ratio of those zones will alter over days of incubation. Thus, due to the expansion in size, necrotic core will get bigger while quiescent zone will thin and shoves towards the very edge of the sphere (Fig. 5A). Increase in size of control spheroids was 2.3 ± 0.1-fold in 8 days (Fig. 5B). Activity on 3-D culture was evaluated for Se-3-AP applied in 3 concentrations (1, 10 and 100 µM). Treatment with 1 µM is not presented due to lack of activity. Se-3-AP at 10 µM caused a delay in growth of MCF-7 spheroids. Regression in size regarding volumes on the day 0 has not been achieved, but those spheres were significantly smaller compared to non-treated controls (Fig. 5B). Considering proportion of the 3 structural zones, there were no major differences observed comparing non-treated and spheres treated with 10 µM (Fig. 5A). On day 6, quiescent zone almost completely merged with the necrotic core barely distinguished on high magnification microscopy (data not shown). Finally, proliferation zone was completely obliterated on day 8, forming a crispy shaped brim as an indication of accelerating cell death. When compared to 10 µM, treatment with Se-3-AP at 100 µM restrained spheroidal growth efficiently (1.0 ± 0.2-fold versus 1.4 ± 0.2-fold computed on day 8 for spheres treated with 100 and 10 µM, respectively). Morphological changes of spheroids in this concentration-treated group were far more striking (Fig. 5A). Starting from day 2, quiescent zone was gradually getting darker, accompanied with progressive contraction of proliferation zone. On day 4, the proliferation zone was barely detectible, while on the next day their edges became shredded. It is interesting to note that necrotic core was easy to distinguish, with the quiescent zone preserved until the end of the treatment.

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Fig. 5

(A) Changes in size and morphology of MCF-7 3-D cultures induced by Se-3-AP applied at concentrations of 10 and 100 µM over the 8-day treatment. Images were acquired daily starting from the day 0 with Celigo imaging cytometer using Celigo software. Scale bar: 200 µm. (B) Growth rates of MCF-7 3-D cultures, non-treated and treated with Se-3-AP applied at concentrations of 10 and 100 µM. Results are expressed as the mean ± SD (n = 3 independent replicates). Statistical significance has been determined by means of Kruskal-Wallis test followed by unpaired t test with Welch’s correction as posttest.

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The SmartFlare technology used here allows us the detection of gene expression in living cells with opportunity for single-cell profiling. In this study, expression levels of octamer-binding transcription factor 4 (Oct-4) was monitored. Oct-4, has been found reactivated in many cancers concomitantly with a reversion of cell phenotype from a highly differentiated state to a highly undifferentiated state, which contributes to cancer development and aggressivity. Knowing the contribution of Oct-4 in development of resistance and tumor recurrence after applied treatment, it is important to monitor its expression so to follow the putative aggressivity of the leftover cells after chemotherapy (Emhemmed et al., 2017). Results of Oct-4 detection should be considered in respect to few important remarks. Assessment of Oct-4 expression has been estimated in the living cells only, a population gated on forward scatter versus side scatter dot plot. Size of this subpopulation varied between from 10 to 20% of all acquired cells in our samples. In terms of previously reviewed results of morphological analysis of non-treated and treated spheroids on day 8, it is obvious that such a low percent of viable cells was due to a massive necrotic core found in control samples, and from accelerated cell death induced by applied treatments. In general, majority of vital cells within spheroids are situated in quiescent zone, consisted of cells in state of dormancy, and in the proliferating zone.

In non-treated spheroids, a high percentage of cells displayed active transcription of Oct-4 gene after 16 h of incubation with SmartFlare detection probe (Fig. 6). Treatment with Se-3-AP induced a noteworthy down-regulation of Oct-4 expressing cells in concentration-dependent manner (Fig. 6A). While 10 µM Se-3-AP abolished Oct-4-transcription in almost 50% of cells, treatment with 100 µM induced a reduction up to 75%. Additionally, according to MFI values, significant decrease in median Oct-4 mRNA transcripts per cell was achieved only in spheroids subjected to Se-3-AP at 100 µM (Fig. 6B). Those results reveal that treatment with 100 µM Se-3-AP stimulates a strong phenotype reprogramming of MCF-7 cells.

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Fig. 6

(A) Histogram of population of cells positive for RNA transcripts of Oct-4 mRNA in MCF-7 3-D cell cultures afterwards 8-day treatment with Se-3-AP applied at 10 and 100 µM (green), and non-treated controls from the same experiment (red). RNA transcription was determined by SmartFlareRNA detection probe. Results are expressed as the mean ± SD (n = 3 independent 3-D replicates). (B) MFI (AU) of Oct-4 expression indicating on median quantity of Oct-4 RNA transcripts per cell. Results are expressed as the mean ± SD (n = 3 independent 3-D replicates). Statistical significance has been determined by Kruskal-Wallis test, with unpaired t test with Welch’s correction as posttest.

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3.3 Inhibition of Cu-mediated DNA damage

In order to compare ability of Se-3-AP and 3-AP in inhibition of Cu(I)-mediated oxidative DNA damage, the assay developed by Battin et al. (2011) was employed. As it can be seen in Fig. 7, both Se-3-AP and 3-AP showed the ability to mitigate oxidative DNA damage caused by hydroxyl radical generated in the Fenton reaction. Agarose gel electrophoresis of pUC19 DNA plasmid alone revealed that almost all DNA was in a supercoiled form (SC), with nicked form (NC) only in traces, while linear form (L) was not evident (Fig. 7, lanes 1). Hydrogen peroxide alone (Fig. 7, lanes 2) and in a combination with 3-AP and Se-3-AP (Fig. 7A lane 3 and Fig. 7B lane 3, respectively) did not induce any damage on plasmid DNA, since proportion of SC and NC forms remained unchanged in respect to native plasmid DNA. However, when plasmid DNA was incubated with Cu(I) and H₂O₂ (reactive mixture), the SC form completely disappeared, while NC became dominant with L form found in traces (Fig. 7, lanes 4). As it can be seen in Fig. 7 (lanes 5–11), when 3-AP or Se-3-AP were added to the reactive mixture, concentration-dependent shifts in ratio of plasmid DNA forms were achieved.

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Fig. 7

Agarose gel showing a reduction in oxidative DNA damage with increasing concentration of 3-AP (A) and Se-3-AP (B). Lanes: (MW) GeneRuler DNA 0.1–10.0 kb ladder mix; (1) plasmid DNA; (2) DNA + H₂O₂; (3) DNA + 3-AP/Se-3-AP + H₂O₂; (4) DNA + Cu(II)/ascorbate + H₂O₂; (5–11) same as lane 4 with increasing concentration of 3-AP/Se-3-AP: 0.1, 1, 5, 10, 50, 100 and 1000 mM, respectively.

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3.4 In vitro metHb formation

To assess the effect of Se-3-AP on in vitro metHb formation compared to 3-AP, a solution of Hb (0.1 mM), obtained from red blood cells (RBC) lysate of healthy human donor, was incubated without (control) and with Se-3-AP and 3-AP (10, 5 and 1 µM) for up to 22 h at 37 °C. The content of metHb in the incubation mixtures was determined by the standard method (Winterbourn and Carrell, 1977). The results are shown in Fig. 8A. MetHb fraction in non-treated controls at 0 min was 2.4%, which is in the range for metHb values determined in healthy individuals (0–3%) (He and Carter, 1992). Results showed that metHb concentration increased time- and dose-dependently in the samples treated with Se-3-AP and 3-AP (Fig. 8A). 3-AP at concentrations 10 and 5 μM induced statistically significant (p < .05) increase of metHb content compared to the control after 60 and 240 min of incubation, respectively. After 22 h of incubation 10 μM concentration of 3-AP induced about three times higher metHb content, compared to the control. In the samples treated with Se-3-AP (5 and 10 µM), statistically significant increase in metHb concentration (p < .05) was reached only after 22 h of incubation. Also, Se-3-AP and 3-AP (1 μM) prevented metHb formation over the first 240 min of incubation period, while only 3-AP (1 μM) after 22 h led to significantly (p < .05) increased metHb formation, compared to the control.

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Fig. 8

MetHb formation induced by 3-AP and Se-3-AP. (A) RBC lysates [total Hb concentration 0.1 mM in 0.1 mM phosphate buffer saline (PBS) pH 7.4] was incubated without (control) and with Se-3-AP or 3-AP (10, 5 and 1 μM). (B) RBC lysates (total Hb concentrations of 1.5 and 0.1 mM in 0.1 mM PBS pH 7.4) were incubated without (control) and with sixty times lower concentration of Se-3-AP or 3-AP (25 μM or 1.67 μM, respectively). Maximum incubation period was 1320 min at 37 °C. Results are means ± S.D. for three experiments.

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Since oxyHb concentration in the blood of healthy individuals is 15–25 times higher than its concentration in this experiment, to assess the metHb formation in circumstances that are more physiological, 1.5 mM of oxyHb was incubated without (control) and with Se-3-AP and 3-AP (25 µM) for up to 22 h at 37 °C. Results of this experiment were compared to those when 0.1 mM oxyHb was incubated without (control) and with Se-3-AP and 3-AP (1.67 µM), thus in both settings molar ratio of oxyHb: Se-3-AP/3-AP was 60: 1 (Fig. 8B). With elevated Hb concentration in the incubation mixture, effects of Se-3-AP and 3-AP were more pronounced and an increase of metHb content after 180 min of incubation was noticed. Both concentrations of 3-AP (25 and 1.67 μM) led to statistically significant (p < .05) rise in metHb content compared to corresponding controls after 120 min, while Se-3-AP (25 μM) achieved significantly higher genesis of metHb only after 22 h of incubation compared to the corresponding control (6.7 ± 0.1% and 3.8 ± 0.5%, respectively). After 22 h of incubation, for all concentrations of Se-3-AP and all molar ratios of oxyHb: Se-3-AP, the obtained metHb content was always below 8.5% (Fig. 8).

3.5 Interaction with HSA

UV/vis and fluorescence spectroscopy were used to study interaction of Se-3-AP with HSA. UV/vis spectra of Se-3-AP with increasing amounts of HSA (Se-3-AP/HSA molar ratios from 1: 0.06 to 1: 0.5) showed a blue λ_max shift (from 373 to 367 nm, Fig. 9A). Interaction between HSA and Se-3-AP lead to changes in the absorption spectra of Se-3-AP upon its binding to HSA. The changes in fluorescence emission spectra of HSA with increasing amount of Se-3-AP (Se-3-AP/HSA molar ratios from 1.5: 1 to 15: 1) are shown in Fig. 9B. The excitation wavelength was 280 nm, at which both Trp214 and Tyr residues emit fluorescence. In spite of the presence of 18 Tyr residues in HSA molecule (He and Carter, 1992), the HSA intrinsic fluorescence is dominated by single Trp214 in subdomain IIA (Lakovicz, 2006). The binding of Se-3-AP to HSA resulted in fluorescence quenching at λ_em (340 nm). In the presence of Se-3-AP, the intrinsic fluorescence decreased in a concentration-dependent manner, suggesting that Se-3-AP interact with HSA. A blue shift in the emission maximum wavelength occurred, indicating that the microenvironment around Trp214 is altered into a more hydrophobic as the HSA–Se-3-AP adduct is formed (Li and Yang, 2015).

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Fig. 9

(A) Changes in UV/vis absorption spectra of Se-3-AP (64 µM in 100 mM PBS pH 7.4) upon addition of HSA in the following molar ratios Se-3-AP/HSA: (a) 1:0.06; (b) 1:0.07; (c) 1:0.08; (d) 1:0.1; (e) 1:0.125; (f) 1:0.17; (g) 1:0.25; (h) 1:0.5. UV/vis absorption spectrum of HSA (4 µM) is represented with a dashed line. (B) Changes in fluorescence emission spectra of HSA (0.125 µM in 100 mM PBS pH 7.4) upon addition of Se-3-AP in the following molar ratios HSA/Se-3-AP: (a) 1:0; (b) 1:1.5; (c) 1:3; (d) 1:4; (e) 1:5; (f) 1:6; (g) 1:7; (h) 1:8; (i) 1:9; (j) 1:10; (k) 1:11; (l) 1:12; (m) 1:13; (n) 1:14; (o) 1:15.

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Fluorescence quenching data were further processed in order to perform more detailed characterization of Se-3-AP–HSA binding. The obtained Stern–Volmer quenching constant (K_sv), the quenching rate constant (k_q), the effective quenching constant for the accessible fluorophores (K_a), the fraction of accessible fluorophore (ƒ_a), thermodynamic binding parameters, association constants (K_b) and number of binding sites (n) of HSA are given in Table 1 and Figs. S12–S15 (Supplementary data). Obtained values for K_sv decrease as temperature increases. The values for K_sv indicate that the quenching mechanism is static. The obtained k_q values are three orders of magnitude higher than the limiting diffusion rate constant of the biomolecule (∼10¹⁰ M⁻¹ s⁻¹), indicating a static type mechanism of fluorophore quenching (Li et al., 2014). The K_a is inversely correlated with temperature, which is in accordance with K_sv temperature dependency. The obtained values for ΔH and ΔS are –5.70 kJ mol^–1 and 88.56 kJ mol^–1, respectively. At all three temperatures obtained values for n were close to 1.

To further examine the binding of Se-3-AP to HSA, the docking in ibuprofen binding site of HSA was performed. Molecular docking gave conformations with the lowest binding energy of –5.5 kcal mol^–1, i.e. 92 µM. There is an aromatic interaction formed between Trp214 and aromatic ring of Se-3-AP. Selenium atom also forms Se⋯π interactions with the same amino acid residue. Hydrogen bonds with Ser464 and Glu450 are formed via selenoamide group (Fig. 10).

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Fig. 10

Se-3-AP docked into ibuprofen binding site of HSA. Green: hydrogen bonds, pink: aromatic interactions; yellow: Se⋯π interactions.

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3.6 Acute lethality assay

Acute toxicity of 3-AP and Se-3-AP was tested on brine shrimp Artemia salina after 24 h incubation and results are expressed as LC₅₀ values. DMSO, as negative control, did not cause changes in viability of treated nauplii. Treatment with K₂Cr₂O₇, as positive control, induced high incidence of lethality (LC₅₀ = 248.14 ± 16.85 μM). Nauplii revealed different level of sensitivity toward 3-AP and Se-3-AP, where LC₅₀ values are 563.41 ± 21.35 and 867.27 ± 19.78 μM, respectively. While LC₅₀ for 3-AP was found to be only 1.5-fold higher compared to LC₅₀ of K₂Cr₂O₇, the concentration of Se-3-AP has to be three times as that of K₂Cr₂O₇ to achieve the death in a half of treated nauplii.