9-Norbornyl-6-chloropurine Is a Novel Antileukemic Compound Interacting with Cellular GSH

Materials and Methods

Materials. NCP, the glutathione conjugate of NCP, NCP-cysteinylglycine and NCP-cysteine were synthesized as described previously (8, 9). 9-[(1R*,2R*,4S*)-Bicyclo[2.2.1][5’_exo,6’_exo-³H₂]hept-2-yl]-6-chloro-9H-purine ([³H]NCP) with specific activity 47.5 Ci/mmol was prepared by the catalytic tritiation of the corresponding 5’,6’-unsaturated precursor at the Laboratory of Radioisotopes (IOCB, Czech Republic). Its chemical identity was confirmed by ³H and ¹H Nuclear magnetic resonance (NMR); specific activity was assayed by a combination of radio-high-performance liquid chromatography and liquid scintillation counting. Allopurinol, ethacrynic acid (GST inhibitors), 1-aminobezotriazole, buthionine sulfoximine (BSO), sulfosalicylic acid, streptomycin, penicillin G, phosphate buffered saline (PBS), RPMI-1640 medium and Minimum Essential Medium Eagle's (Eagle's MEM) were purchased from Sigma–Aldrich (St. Louis, MO, USA); fetal calf serum was obtained from PAA Laboratories GmbH (Pasching, Austria). Subcellular fractions from rat liver homogenate were isolated using previously published procedures (10). Soluene 350® was from PerkinElmer (Waltham, MA, USA), salts for buffer preparations and NADPH were from Serva (Heidelberg, Germany). HUVEC-2 cells, Endothelial Cell Culture Medium, BD BioCoat™ HTS Caco-2 Assay System, Seeding Basal Medium™ and Enterocyte Differentiation MediumTM were from BD Biosciences (Heidelberg, Germany). All other cell lines and Fibroblast Basal Medium were from LGC standard (Teddington, UK).

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Figure 1.

Structure of 9-[(1R*,2R*,4S*)-bicyclo[2.2.1]hept-2-yl]-6-chloro-9H-purine (9-norbornyl-6-chloropurine, NCP).

Cell culture. CCRF-CEM, HL-60, HeLa-S3, HepG2, HUVEC-2, NHDF-Ad and Caco-2 cells were cultured under a humidified atmosphere containing 5% CO₂ at 37°C. CCRF-CEM, HL-60, HeLa-S3 and HepG2 cells were grown in RPMI-1640 or Eagle's MEM supplemented with 10% or 20% (Caco-2) fetal calf serum, 200 mg/ml of streptomycin, 200 U/ml of penicillin G and 4 mM glutamine. HUVEC-2 cells were grown in Endothelial Cell Culture Medium. NHDF-Ad cells were grown in Fibroblast Basal Medium. 1% Non-essential amino acid solution, 1 mM sodium pyruvate and 1.5 g/l NaHCO₃ was added to the medium for Caco-2 cells.

Preparation of crude cellular extract. Cells (1.5×10⁹) were washed with PBS and suspended in 50 mM Tris–HCl buffer, pH 7.4 containing 1 mM dithiothreitol (DTT), 5 mM MgCl₂, protease inhibitor cocktail (Sigma–Aldrich) and 0.4% NP-40. The cells were sonicated for 5 s three-times and homogenized in a Dounce homogenizer. The homogenate was centrifuged at 30,000 ×g for 30 min. The supernatant was incubated with streptomycin sulfate for 1 h to remove nucleic acids and centrifuged at 30,000 ×g for 30 min. The resulting crude cell extract was desalted on PD-10 columns (GE Healthcare, Freiburg, Germany), aliquoted and stored at −80°C. The protein content was determined by the BCA kit (Sigma–Aldrich), according to the manufacturer's instructions.

Non-radioactive in vitro metabolic assays and HPLC analysis of the metabolites. NCP (100 μM) was incubated with crude CCRF-CEM cell extract (5.4 mg of protein per ml) with/without the inhibitors of xanthine oxidase, GST and cytochrome P450 (0.1 and 1 mM allopurinol, 0.05 and 1 mM ethacrynic acid and 0.1 and 1 mM 1-aminobenzotriazole, respectively) in 0.05 mM phosphate buffer, pH 7.4, for 30 min at 37°C. For the study of the inhibition of cytochrome P450, NADPH (1 mM) was included in the reaction. The reactions were stopped and extracted with 100% methanol and chilled at −20°C for 1 h. Then the samples were centrifuged at 8,000 ×g for 2 min in centrifugal filter units (Ultrafree-MC dura 0.22 μm, Millipore, Billerica, Massachusetts, USA). The filtrates (50 μl) were injected onto a Supelcosil LC-18-S HPLC column (150 mm × 4.6 mm I. D., 5 μm; Sigma Aldrich) and eluted in a non-linear gradient of 10-30% acetonitrile in 50 mM phosphate buffer. The flow rate was 0.9 ml/min. The absorption spectra of the eluate were recorded by a Photodiode array detector (PDA; Waters Corporation, Milford Massachusetts, USA). The data were analyzed at 260 nm.

[³H]NCP metabolism whole CCRF-CEM cells and in vitro. CCRF-CEM cells (5×10⁵ cells/ml) were preincubated with or without 50 μM BSO in a CO₂-incubator at 37°C. After 24 h, [³H]NCP was added to a final concentration of 25 μM (10 μCi/ml). After another 24 h of incubation, the cells were washed with PBS and pelleted by centrifugation at 250 ×g for 5 min. The pellets were extracted with 100 μl of 70% methanol and chilled at −20°C for 1 h. The equivalents of RPMI-1640 medium (200 μl) were extracted with 583 μl of 100% methanol and chilled at −20°C for 1 h. Alternatively, crude CCRF-CEM cell extract (5.4 mg of protein per ml) or rat liver subcellular fractions (protein content 4.6-5.4 mg/ml) were incubated with 25 μM (1 μCi/ml) NCP in 0.05 mM phosphate buffer, pH 7.4 for 30 min at 37°C. Where indicated, 1 mM ethacrynic acid was added. The reactions were extracted with 100% methanol and chilled as described above. The HPLC analysis was carried out as described in the previous section. Fractions of 0.23-ml of the HPLC-eluate were collected and the radioactivity was counted in Aquasafe® scintillation cocktail (Zinsser Analytic, Maidenhead, UK). Radioactivity (CPM) was plotted against retention time to obtain a high-sensitivity [³H]NCP metabolite profile.

Glutathione (GSH) determination. Cells preserved in sulfosalicylic acid were thawed, sonicated for 5 min on ice, centrifuged for 5 min at 5,000 ×g and the supernatants were used for the assay. Total GSH content was determined using the enzymatic recycling method with 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB) and GSH reductase in a microplate format. The rate of TNB formation was recorded at 405 nm for 2 min and the slope was compared to that of the standard curve of GSH disulfide (GSSG). The concentration of total GSH was calculated using the method of linear regression. The results were then protein-correlated and expressed as relative values, taking the total GSH in control sample as 100%.

Cytotoxicity evaluation. The cytotoxicity of the tested compounds was assessed with the XTT sodium salt cell proliferation kit II (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. CCRF-CEM, HL-60, HeLa-S3, HepG2, HUVEC-2 and NHDF-Ad cells were seeded in a 96-well plate at a density of 13,500, 18,000, 2,700, 19,800, 4,000 and 4,000 cells per well, respectively. After 24 h, the tested compounds were added to the culture media and incubated for 72 h before the XTT dye was added. The absorbance at 495 nm was recorded after a 1-h incubation with the dye. Half-maximal inhibitory concentration (IC₅₀) values were determined by GraphPad Prism version 5.00 for Windows (GraphPad Software, La Jolla, CA, USA). Where indicated, cells were preincubated with 50 μM BSO for 24 h prior to the addition of NCP.

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Figure 2.

High-performance liquid chromatography analysis of intra- and extracellular [³H]9-norbornyl-6-chloropurine [(³H]NCP)] metabolites. CCRF-CEM cells were incubated with 25 μM (10 μCi/ml) [³H]NCP (−) for 24 h. In a parallel experiment, CCRF-CEM cells were preincubated with 50 μM buthionine sulfoximine for 24 h and after that 25 μM (10 μCi/ml) [³H]NCP was added (⋯). The identity of all peaks in the cells (A) and the medium (B) was confirmed using the authentic standards. NCP-GS, Glutathione conjugate; NCP-Cys-Gly, NCP-cysteinylglycine; NCP-Cys, NCP-cysteine.

Transepithelial transport of NCP in Caco-2 monolayers. The transport of NCP across Caco-2 monolayers was studied using the BD BioCoat™ HTS Caco-2 Assay System according to the manufacturer's instructions. Immediately prior to the transport experiments, the monolayers were washed twice with HBSS containing 10 mM HEPES and 25 mM glucose, pH 7.4 and incubated for 30 min in a CO₂-incubator at 37°C. A total of 2×10⁵ cells in Seeding Basal Medium™ were seeded in each transwell insert, left to attach for 48 h and the medium was then changed for Enterocyte Differentiation Medium™and cultured for the next 48 h. The integrity of cell monolayers was assessed by monitoring the transepithelial electrical resistance (TEER) using a Millicel ERS apparatus (Millipore) and paracellular flux marker, [¹⁴C]mannitol (0.15 μCi/insert). Intact cell monolayers displaying TEER higher than 250 Ω cm² were used for the experiments. The transport was initiated by the addition of 1 ml of the transport medium to the basolateral side and 300 μl of the transport medium containing 10 μM [³H]NCP (0.15 μCi), to the apical side. After a 3-h incubation, 80 μl aliquots were collected from the donor and acceptor sides. The radioactivity was counted in Aquasafe® scintillation cocktail. The apparent permeability coefficient (P_app) was calculated from the following formula: P_app=(dQ/dt)/C₀×A where dQ/dt is the rate of absorption of the drug across the cells, C₀ is the donor compartment concentration at time zero and A is the area of the monolayer.

Data analysis and statistical procedures. Unless otherwise indicated, the data are presented as the mean±SD from at least three independent experiments. Statistical evaluation was performed using GraphPad Prism version 5.00 for Windows (GraphPad Software).