(−)-Epicatechin Enhances Etoposide-induced Antileukaemic Effect in Rats with Acute Myeloid Leukaemia

Materials and Methods

Chemicals. Reagents including EC, propidium iodide (PI), red blood cell lysing buffer with ammonium chloride, polyethylene glycol, polysorbate 80, benzyl alcohol, citric acid, ethanol and all reagents used in ferric ion reducing ability of plasma (FRAP), malondialdehyde (MDA) and superoxide dismutase (SOD) investigation were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA). VEPESID (etoposide) was purchased from Bristol-Myers Squibb S.p.A. (Sermoneta, Italy). RPMI 1640 medium, fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from PAA (Pasching, Austria). Fluorescein isothiocyanate (FITC)-conjugated mouse anti-rat CD45R monoclonal antibody, annexin-V-allophycocyanin (APC) and 7-aminoactinomycin D (7-AAD) were obtained from BD Biosciences Pharmingen (San Diego, CA, USA). (PE)-conjugated goat anti-mouse antibody specific against μ chain was acquired from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Animals. Male Brown Norway (BN/CrlCmd) rats (297.2±3.6 g) were obtained from the Animal Center, Polish Academy of Sciences Medical Research Center (Warsaw, Poland). The animals were housed in plastic cages and kept under standard conditions of 22°C and 50-60% humidity, with a 12-h light-dark cycle. They were fed a synthetic pellet diet (Morawski Label Feed, Kcynia, Poland) and had free access to water.

BN rat myeloid leukaemia model. BN rats with acute myeloid leukaemia (BNML) are regarded as one of the best animal models of myeloid leukaemia due to their many similarities to human AML, e.g. growth pattern (23). In this experimental model, leukaemia is transplantable and BNML cells are inoculated into immunologically competent rats. In the present study, BNML was developed after intravenous inoculation with 10⁶ spleen-derived leukaemia cells (kindly provided by Professor A.C.M. Martens, Utrecht University, the Netherlands) in PBS. Experiments were performed in accordance with legal requirements, under a licence granted by the Jagiellonian University Ethical Committee.

Treatment protocol. The doses of EC (20 and 40 mg/kg b.w.) were chosen based on our previous research in which EC at a dose of 40 mg/kg b.w. significantly increased apoptosis, while a lower dose (20 mg/kg b.w.) did not induce such an effect on BNML cells of leukaemic rats (16). EC was dissolved in hot water and, after cooling, was administered by oral gavage (40 mg/kg b.w. in 0.5 ml of water) from the second day after the inoculation of BNML cells, every 24 hours until day 23 of leukaemia development. In the study, 50 mg of etoposide/kg b.w. of rat was used, which induced severe DNA damage in the bone marrow of healthy mice (24). Etoposide was administered every 24 hours for three consecutive days starting on the 21st day of leukaemia development. Etoposide was administered intraperitoneally to avoid possible influence of EC on its intestinal bioavailability. Control rats were administered with solvents of investigated compounds. Six to eight leukemic rats were included in each group. Six healthy control and EC-treated rats were also used to investigate the influence of EC on the oxidative status. These rats were killed 1.5 hour after etoposide and 2 hours after EC administration. Turner et al. (24) observed the greatest DNA damage in the bone marrow of mice one hour after peritoneal administration of etoposide. Behaviour, weight and general condition of the animals were monitored.

Organ excision and cell isolation. Rats were killed by cervical dislocation under vetbutal anaesthesia. The right femurs were removed and the bone marrow was used for the preparation of smears using a small paintbrush. Bone marrow from left femurs was flushed with 1 ml FBS and then washed once in PBS (230 ×g, 10 min, 4°C). Spleens were excised, weighed and macerated into a Petri dish filled with RPMI-1640 medium, and subsequently centrifuged (230 ×g, 10 min, 4°C). Livers were excised, immediately frozen and stored at −80°C until redox state investigation. In order to examine cells by flow cytometry, the isolated cells were counted in a Bürker haemocytometer and erythrocytes were lysed with red blood cell-lysing buffer containing 0.83% ammonium chloride in 0.01 M Tris buffer (pH 7.5).

Cytological study of bone marrow. Smears were stained using May-Grünwald-Giemsa stain and examined by counting approximately 500 cells/slides on three slides from each animal using an Olympus CX-40 microscope (Olympus, Tokyo, Japan).

Detection of BNML cells and analysis of apoptotic and necrotic cell death. Isolated cells (1×10⁶) were incubated for 45 min on ice in 100-μl PBS containing 5% FBS and 10% mouse primary monoclonal anti-RM124 antibody (kindly provided by Professor A.C.M. Martens), which recognises the specific epitope of the leukaemia cells (23). After incubation, the cells were washed in PBS (230 ×g, 10 min, 4°C). Next, they were incubated with secondary goat anti-mouse monoclonal antibody conjugated to R-PE for 30 min and washed in PBS (230 ×g, 10 min, 4°C). The cells were than incubated with FITC-conjugated mouse anti-rat CD45R monoclonal antibody for 20 min to evaluate the nonspecific binding of the secondary antibody to B-lymphocytes. Subsequently, the cells were stained with annexin V-APC (An-APC) and 7AAD according to the instructions provided by the manufacturer and incubated in the dark for 15 min. All the steps were performed on ice. The cells were analyzed using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences Immunocytometry Systems, San Jose, CA, USA). Apoptotic cells were defined as APC-An-positive and 7AAD-negative, while necrotic and/or late apoptotic cells were APC-An- and 7AAD-positive, necrotic cells were 7AAD-positive and APC-An-negative. Results were expressed as the percentage of apoptotic or necrotic cells.

Measurement of FRAP. The method of Benzie and Strain (25) was used to determine FRAP as an indicator of the antioxidant content. FRAP values were obtained by measuring ferric to ferrous ion reduction at low pH (300 mmol acetate buffer, pH 3.6), coupled with tripyridyltriazine, a blue-colored complex, whose absorbance was measured at 593 nm. Tissues were homogenized in 0.05 M phosphate buffer (pH 7.8) to 10% final concentration. Homogenized tissues were centrifuged at 1000 ×g for 15 min (0-4°C). The supernatant was drawn and the pellet was discarded. Each sample (0.1 ml) was mixed with 3 ml reagent mixture [30 ml, consisting of 25 ml of acetate buffer (300 mM; pH 3.6), 2.5 ml 2,4,6-Tris(2-pyridyl)-S-triazine (5 mM TPTZ) in 40 mM HCl, and 2.5 ml ferric solution (20 mM FeCl₃) in distilled water]. The FRAP was calculated by preparing a standard curve between 0 and 1 mM of known Fe²⁺ sample using FeSO4•7H₂O as the standard. The blank contained FRAP reagent mixture. Reactions were performed for 5 min at 37°C and analyzed spectrophotometrically. FRAP values are expressed as μmol/mg of protein.

Investigation of lipid peroxidation. The concentration of MDA, a product of lipid peroxidation, was measured in liver homogenates according to the method of Buege and Aust (26), based on thiobarbituric acid (TBA) reactivity. Reaction of MDA with TBA followed by measurement of the pink chromophore product was used for quantitative determination of MDA. Each sample (0.5 ml) was mixed with 1.5 ml reagent mixture (TBA/TCA/HCl reagent by diluting the stock solution 4-fold in water) to maintain a reagent/sample ratio of 2:1 (v/v). Working solution was prepared by diluting the stock solution of TBA/TCA/HCl 4-fold in water. Butylated hydroxytoluene (BHT) in ethanol was added to a final concentration of 0.03%. After shaking the tube, the mixture was placed into boiling water bath for 15 min. After cooling in cold water, the obtained chromogen was extracted with 3.0-ml n-butyl alcohol by vigorous shaking for 1 min. Separation of the organic phase was facilitated by centrifugation at 1000 ×g for 10 min. The TBA-MDA adduct was quantified using fluorescence emission at 553 nm upon assessment of cell toxicity excitation at 532 nm. The MDA concentration was measured by preparing a standard curve between 0 and 50 nmol MDA/sample using 1,1,3,3-tetramethoxypropane as the standard. MDA concentrations are expressed as nmol/mg of protein. The measurements were performed by using a spectrofluorometer (Perkin Elmer LS-5, USA).

Measurements of SOD activity. SOD activity was investigated by the method of Spitz and Oberley (27) in homogenates from liver. Xanthine–xanthine oxidase was utilized to generate a superoxide flux. Nitro-blue tetrazolium (NBT) reduction by O₂^•− to blue formazan was followed at 560 nm in a Marcel Media (Poland) spectrophotometer at room temperature. The rate of NBT reduction in the absence of tissue was used as the reference rate (0.020±0.005 AU/min). When increasing amounts of protein (containing SOD activity) were added to the system, the NBT reduction rate was progressively inhibited. The amount of inhibition was defined as a percentage of the reference rate of NBT reduction, when SOD activity was absent. The data were plotted as the percentage inhibition vs. protein concentration. One unit of activity was defined as the amount of protein necessary to reduce the reference rate to 50% of maximum inhibition. The assay mixture contained catalase to remove H₂O₂ and diethylenetriaminepenta-acetic acid (DETAPAC) to chelate metal ions capable of redox cycling and interference with the assay system. The measurements were performed spectrophotometrically using a Marcel Media spectrophotometer (Poland).

Tissues were homogenized in 0.05 M phosphate buffer (pH 7.8) to a final concentration of 10%. Homogenates were sonicated and then centrifuged at 1000 ×g for 15 min (0-4°C). The supernatant was drawn and the pellet was discarded. Each supernatant (0.1 ml) was mixed with 0.8 ml reagent mixture containing: bovine serum albumin (BSA) in DETAPAC buffer, 50 mM potassium phosphate monobasic and dibasic buffer, (pH 7.8); 1 mM DETAPAC; 0.13 mg/ml BSA (12.90 ml); 1.0 U bovine liver catalase (0.5 ml); 5.6×10⁻⁵ M NBT (0.5 ml); 0.1mM xanthine (1.7 ml); 0.05 M potassium phosphate buffer (0.3 ml); 50.0 μM bathocuproine disulfonate disodium salt (BCS) (0.1 ml) and 0.1 ml of the xanthine oxidase, 13.2 U/ml to achieve the required reference rate. All data were expressed in units of SOD activity per milligram of protein.

Statistical analysis. One-way analysis of variance ANOVA and post-hoc Tukey test were used to calculate the statistical significance between groups. Values of SOD activity were assessed by the nonparametric Mann-Whitney test. Data are represented as the mean±standard error of the mean.