Thiazolyl blue Tetrazolium bromide (MTT), verapamil, fetal bovine serum (FBS), 5,5′-dimethyl-oxazolidine-2,4-dione (DMO), cyclosporin A (CsA), nicotinamide adenine dinucleotide reduced form (NADH), nicotinamide adenine dinucleotide phosphate reduced form (NADPH), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), 3,4-dimethoxybenzoic acid, hydrochloric acid, acetonitrile and sodium bicarbonate were purchased from Sigma-Aldrich. All cell culture flasks and dishes were obtained from Corning, Inc. AGE and SAC provided by Wakunaga Pharmaceutical Co. Ltd. (Hiroshima, Japan) were manufactured as follows: Garlic cloves were sliced, immersed in a water-ethanol mixture solution and naturally extracted for >10 months at room temperature, as previously described (28). The AGE powder used in our experiments was prepared by lyophilization. It contained approximately 28.6% (w/v, 286 mg/ml) solid material, 0.63% (6.3 mg/ml) arginine and 0.1% SAC (calculated on a dry weight basis) as a marker compound for standardization (29). Both SAC and the AGE powder was freshly dissolved in complete Ham's F-12 or in RPMI-1640 medium prior to each experiment. The analysis of the organosulfur compound content in the AGE powder was performed using a Shimadzu LC-6A HPLC system. The content of SAC was determined by ion-exchange chromatography, using a column TSK-GEL Amino Pak, followed by post-derivatization using o-phthalaldehyde (OPA), and a fluorescent detection with excitation at 340 nm and emission at 455 nm. The test samples for the analysis of SAC were prepared by the addition of 5% trichloroacetic acid, as previously described (30). The SAC powder was freshly dissolved in RPMI-1640 medium supplemented with 10% FBS, normally up to 50 mg/ml, prior to each experiment. During the experiment the solution, which was kept in the dark, was for one use only.

Two types of NB cells were used in this study. One was the SJ-N-KP cells, a non-amplified NB cell line and the second one was the IMR5 cells, a n-myc amplified NB cell line (31). Both cell lines were a kind gift from Dr N. Crescenzio (Department of Paediatrics, University of Turin, Regina Margherita Children's Hospital, Turin, Italy) and Dr F. Timeus (Paediatric Haematology-Oncology, Regina Margherita Children's Hospital) (31). The NB cell lines, SJ-N-KP and IMR5 were maintained in monolayer cultures in RPMI-1640 medium supplemented with 10% FBS, 2 mM L-glutamine, 100 µg/ml streptomycin and 100 IU/ml penicillin (31). Both cell lines were incubated in a humidified atmosphere of 5% CO₂ in a water-jacketed incubator at 37°C. For each passage, exponentially growing SJ-N-KP and IMR5 cells were harvested with 10 mM EDTA and by the further addition of 0.10% trypsin solution. The trypsin activity was quenched by the addition of complete RPMI-1640 medium (31).

To detect phosphatidylserine residue exposure on the surface of the plasma membrane of the tumor cells in the initial step of apoptosis, an Annexin V-FITC apoptosis detection kit was used as previously described by Van Engeland et al (32). The NB cell lines, SJ-N-KP and IMR5 (9.1×10⁴ cells/ml), were seeded in a 6-well plate, containing complete RPMI-1640 medium and incubated for 24 h at 37°C to allow for the the complete reattachment of the cells to the plates. After exchanging the medium with fresh medium, the cells were incubated at 37°C in the presence of 0, 10 and 20 mM SAC for 48 h. Following incubation at 37°C, the cells were detached, washed with PBS, centrifuged at 400 × g for 2 min at 25°C and then stained with 1 µg/ml of Annexin V-FITC and with 1 µg/ml of PI for 10 min at room temperature in the dark. Annexin V-FITC and PI fluorescence were measured on the FL-1 channel (533/30 nm) and the FL-3 channel (>670 nm), respectively, with excitation at 488 nm. A minimum of 10,000 events/sample was acquired (32).

Cell cycle distribution was analyzed by labeling the cells with PI. The assays were carried out as previously described by Nicoletti et al (33). S-allyl-L-cysteine-treated and the untreated NB cell lines, SJ-N-KP and IMR5 (9.1×10⁴ cells/ml), as described above in Annexin V-FITC labeling, were harvested, washed twice with cold PBS and centrifuged at 400 × g for 2 min at 4°C. The pellet was fixed in 70% ethanol at −20°C for 1 h. After washing twice with PBS, the cells were resuspended in PBS containing 100 µg/ml RNase A and 40 µg/ml PI. Following incubation at 37°C for 1 h, the cells were subsequently analyzed by flow cytometry using the FL-3 channel (>670 nm) with the acquisition of 10,000 events/sample.

The NB cell lines, SJ-N-KP and IMR5, were seeded in a 96-well plate and incubated for 24 h to allow for the complete reattachment of the cells to the plates. After the medium was replaced with fresh medium, the cells were incubated in the presence of 0, 1, 5, 10, 20 and 30 mM SAC for 48 h. The anti-proliferative effects of SAC on the human NB cells were examined by MTT assay. Briefly, MTT was added to each well at a final concentration of 0.5 mg/ml. Following 3 h of incubation at 37°C, dimethyl sulfoxide was added to dissolve the crystals. The absorbance was determined at 577 and 660 nm using a spectrophotometer multi-mode plate reader [Synergy HT BioTek, serial no. 270204; BioTek, Bernareggio (MB)].

The changes in Δψm in whole cells were assayed using the lipophilic cationic probe, JC-1 dye. The NB cell lines, SJ-N-KP and IMR5, were seeded in a 12-well plate and incubated for 24 h at 37°C to allow the cells to adhere to the plates. After the medium was replaced with fresh medium, the cells were incubated with 30 and 50 mM of SAC for 24 h at 37°C. Subsequently, the cells were stained with 2.5 µg/ml of JC-1 for 20 min at 37°C. The detached cells were washed with PBS and then resuspended in PBS. The samples were then analyzed using a BD Accuri C6 flow cytometer (BD Biosciences). JC-1 was excited using an argon laser at a wavelength of 488 nm (using a BD Accuri C6 flow cytometer). The emitted green (JC-1 monomer) and red (JC-1 aggregate) fluorescence were detected at the FL-1 channel (533/30 nm) and FL-2 channel (585/40 nm), respectively. At least 10,000 events/sample were acquired in log mode. The ratio of red (FL2)/green (FL1) fluorescence intensity was used to represent the Δψm.

SAC was analyzed by GC-MS as its tert-butyldimethylsilyl (TBDMS) derivative according to Jiménez-Martín et al (34). Briefly, a 20 µl aliquot of a solution containing SAC at 1 mg/ml in 0.1 N HCl spiked with 15 µl internal standard (3,4-dimethoxybenzoic acid, 0.1 mg/ml) was dried, and 50 µl of neat N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA), followed by 50 µl of acetonitrile, were added. The mixture was heated at 100°C for 2 h. The sample was then neutralized with sodium bicarbonate and subjected to GC-MS analysis. The same derivatization protocol was used for AGE power (1 mg/0.1 M HCl). GC-MS analyses were performed with an Agilent 7890B gas chromatograph coupled to a 5977B quadrupole mass selective detector (Agilent Technologies). Chromatographic separations were carried out with an Agilent HP5 ms fused-silica capillary column (30 m × 0.25 mm i.d.) coated with 5%-phenyl-95%-dimethylpolysiloxane (film thickness, 0.25 µm) as a stationary phase. The injection mode was splitless at a temperature of 280°C, the column temperature program was 70°C (1 min) then to 300°C at a rate of 20°C/min and held for 10 min. The carrier gas was helium at a constant flow of 1.0 ml/min. The spectra were obtained in the electron impact mode at 70 eV ionization energy; ion source 280°C; ion source vacuum 10⁻⁵ Torr. MS analysis was performed simultaneously in TIC (mass range scan from m/z 50 to 600 at a rate of 0.42 scans/sec) and SIM mode. GC-SIM-MS analysis was performed selecting the following ions: m/z 302 and m/z 332 for SAC and m/z 239 for 3,4-dimethoxybenzoic acid (internal standard).

A total of 50 male Wistar rats, 2 months old, weighing approximately 150 g, were used in our experiments. The rats, housed in the animal facility of the Department of Biomedical Sciences, University of Padova, were maintained under controlled conditions (temperature 20–22°C, relative humidity 48–50%, water with antibacterial control and a 12:12 h light/dark cycle) and provided with water and a standard diet (4RF25) purchased by Mucedola s.r.l., Settimo Milanese (MI) according to Ohkubo et al (1). The experimental procedures were approved by the local Ethics Committee for Animal Experimentation (CEASA) (protocol no. 3619, 15.1.2014) and performed in agreement with the international guidelines, as well as European Communities Council Directive and National Regulations (CEE Council 86/609 and DL 116/92).

Rat liver mitochondria (RLM) were isolated by the differential centrifugation method according to Ohkubo et al (1). In detail, the rats were starved overnight and sacrificed by cervical dislocation. The livers were rapidly explanted, immersed in ice-cold isolation medium containing 250 mM sucrose, 5 mM HEPES (pH 7.4), 0.5 mM EGTA and washed 4/5 times with the same medium. The livers were minced into small sections and washed with ice-cold fresh medium without EGTA. The suspension was transferred to a glass potter and homogenized using a Teflon pestle operating at 1,600 rpm, by 3–4 strokes. The homogenate was centrifuged at 700 × g for 5 min at 4°C and the obtained supernatant was centrifuged at 10,800 × g for 10 min at 4°C. The pellet was washed with isolation medium, resuspended and centrifuged at 15,900 × g for 10 min at 4°C. Finally, the obtained pellet, containing the mitochondria, was suspended in the standard medium (see the incubation procedure) (35). Mitochondrial proteins were measured by the biuret method with BSA, as a standard (36). The mitochondria (1 mg protein/ml) were incubated in a water-jacketed cell at 25°C under continuous stirring. The standard medium contained 250 mM sucrose, 10 mM HEPES (pH 7.4), 5 mM Na-succinate, 1.25 µm rotenone and 1 mM Na-phosphate. Variations and/or other additions are provided with each experiment.

Mitochondrial membrane potential (ΔΨ_m) was calculated by detecting the distribution of the lipid-soluble cation tetraphenylphosphonium (TPP⁺, Sigma-Aldrich) through the inner membrane, measured by a TPP⁺ selective electrode, according to Kamo et al (37). The mitochondrial electrochemical gradient (58ΔpH_m) was calculated from the distribution of DMO ([¹⁴C]5,5′-dimethyl-oxazolidine-2,4-dione) across the mitochondrial membrane, according to Rottemberg (38). 58 is the value in mV of RT/zF for giving the ΔpH value in mV. The redox state of mitochondrial glutathione was detected by using the Ellman reagent, according to Tietze (39). The change with time of the redox state of mitochondrial pyridine nucleotides was followed fluorometrically in an Aminco Bowman 4–8202 spectrofluometer with excitation at 354 nm and emission at 462 nm.

The data were presented as the means ± SEM or means ± SD. Statistical analysis was performed using one-way ANOVA with the Dunnett's post hoc test, in EZR (Kanda) (40). A P-value <0.05 was considered to indicate a statistically significant difference.

Fig. 1 shows the effects of AGE on the components of the electrochemical gradient (Δµ_H⁺), membrane potential, ΔΨ_m, and the chemical gradient, ΔpH_m, expressed in mV. The results revealed that under normal conditions, both the ΔΨ_m and 58ΔpH_m exhibited the physiological values of 170 and 40 mV, respectively. The presence of AGE induced an initial increase in ΔΨ_m of approximately 30 mV and a corresponding identical decrease in 58ΔpH_m. However, after approximately 15 min, the ΔΨ_m gradually decreased until 120 mV. In addition, the 58ΔpH_m value underwent a parallel drop of approximately 10 mV. The membrane alterations of RLM by AGE are shown in Fig. 1. The decrease in both ΔΨ_m and 58ΔpH_m was accompanied by an increase in the oxidation of endogenous glutathione content. This is evidenced by the decrease in the reduced content of glutathione induced by different concentration of AGE as reported in Fig. 2. AGE also caused the oxidation of endogenous pyridine nucleotides, NADH and NADPH, as observed by the downward deflection of the curves detecting the reduced state of these nucleotides in Fig. 3. It is also to evidence that cyclosporine A (CsA) strongly inhibits the oxidation of both demetabolites.

In this study, we evaluated the use of the silylation reagent, MTBSTFA, for the derivatization of SAC. MTBSTFA derivatives are more stable and less moisture-sensitive than those formed using lower molecular weight reagents. A chromatogram of SAC is presented in Fig. 4. In the chromatographic condition described above, SAC has a retention time of 9,8 min and an electron impact mass spectrum (Fig. 4, inset) showing a typical fragment (m/z 302) corresponding to the molecular weight of the derivative less C₃H₅S (M-73). In Fig. 5, a typical GC-MS chromatogram of AGE is presented. The chromatogram shows the presence of a peak having the same retention time and mass spectrum of SAC. In addition, TBDMS derivatization allows for the identification of in AGE a number of polar compounds, including amino acids and carboxylic acids. Amino acid derivatives were identified by comparing spectral data with those reported in the NIST2018 library. Electron impact spectra of these derivatives contain typical fragments corresponding to the molecular weight of the derivative less CH₃ (M-15), C₄H₉ (M-57), C₄H₉ + CO (M-85) and CO-O-TBDMS (M-159).

The results of MTT assays revealed that treatment with SAC exerted anti-proliferative effects on the NB cancer cells examined in a dose-dependent manner. Treatment with 20 mM of SAC for 48 h decreased the viability of both the SJ-N-KP and IMR-5 cells to 30.05 and 33.58%, respectively, when compared with the untreated control cells (Fig. 6). The proliferation of both cell lines was already markedly decreased to approximately 63.77% of the control level following treatment with 5 mM SAC for the same incubation time of 48 h (Fig. 6). A further extension of the incubation time at 72 h, did not considerably affect the viability of the cells (data not shown). A concentration of <5 mM SAC exerted a modest effect of about 10% only on the IMR-5 cells.

The loss of phospholipid asymmetry and the appearance of phosphatidylserine residues on the outer layer of the plasma membrane is an early signal of cell death, namely apoptosis. Annexin V is a Ca²⁺-dependent phospholipid-binding protein with a high affinity for phosphatidylserine. In this study, to detect externalized phosphatidylserine, Annexin V-FITC/PI staining was performed in the NB cell lines, SJ-N-KP and IMR5. The results of flow cytometric analysis are presented in Fig. 7. Treatment with 10 and 20 mM of SAC increased the percentage of the total apoptotic cells (Fig. 7A, middle and upper right panels) to 18.1 and 42.6%, respectively, compared with the untreated SJ-N-KP cells (5.2%) (Fig. 7A, upper left panel). On the contrary, the percentage of apoptotic cells was higher in the IMR5 cells than the SJ-N-KP cells following treatment with 10 and 20 mM of SAC (Fig. 7A, lower panels, 33.0 and 51.7%, respectively, and Fig. 7B), compared with the untreated IMR5 cells (6.2%) (Fig. 7A, lower left panel).

To confirm the involvement of apoptotic cell death induced by SAC, flow cytometric analysis using PI staining was performed to analyze the cell cycle status. The apoptotic cells that undergo DNA fragmentation exhibit sub-G1 DNA contents. The exposure of the NB cell lines, SJ-N-KP and IMR5, to 10 and 20 mM of SAC for 48 h induced a significant increase in the sub-G1 apoptotic cell population, compared to that observed in the untreated control cells (Fig. 8). The percentage of treated IMR5 cells in the sub-G1 phase (38.9 and 67,7%) was higher than the percentage of SJ-N-KP cells in the same phase (24.0 and 44.8%), at both concentrations of SAC, which was consistent with the results of the Annexin V-FITC/PI staining assay (Fig. 7). These results confirm the cytotoxicity induced by both concentrations (10 and 20 mM) of SAC and suggest the involvement of an apoptotic mechanism.

To investigate the mechanisms through which SAC induces cell death, we examined the loss of Δψm using a flow cytometric analysis of the control and treated cells loaded with the mitochondrial probe, JC-1. In healthy cells, membrane-permeable JC-1 dye spontaneously accumulates in the mitochondria and forms aggregates known as J-aggregates that emit red fluorescence after excitation. By contrast, in apoptotic cells, with a low Δψm, JC-1 remains in the cytoplasm as monomers, which emit green fluorescence. Therefore, the loss of Δψm is indicated by a decrease in the ratio of red/green fluorescence intensity. As shown in Fig. 9, in both the NB cell lines SJ-N-KP and IMR5, exposure to SAC induced an evident MMD. Treatment with SAC increased the ratio green/red fluorescence intensity and concomitantly decreased the red fluorescence intensity in a dose-dependent manner. The SAC-induced Δψm dissipation was more evident in the IMR5 cells than the SJ-N-KP cells following treatment with 50 mM SAC solutions for 48 h at 37°C (average 46.6 and 59.1%, respectively). Moreover, a decrease in MMD was already observed in the IMR5 cells at lower concentrations of SAC, 30 mM, whereas the same concentration of SAC did not induce any variations in the SJ-N-KP cells, as shown in Fig. 9.