Biological Activity of Hydantoin Derivatives on P-Glycoprotein (ABCB1) of Mouse Lymphoma Cells

Materials and Methods

Compounds. Thirty hydantoin derivatives (SZ-2, SZ-7, LL-9, BS-1, JH-63, MN-3, TD-7k, GG-5k, P3, P7, P10, P11, RW-15b, AD-26, RW-13, AD-29, KF-2, PDPH-3, Mor-1, KK-XV, Thioam-1, JHF-1, JHC-2, JHP-1, Fur-2, GL-1, GL-7, GL-14, GL-16, GL-18) were tested, kindly provided by Dr. Jadwiga Handzlik and Prof. Dr. Katarzyna Kieć-Kononowicz, Cracow, Poland). The compounds were dissolved in DMSO.

Other chemicals used in the study were: doxorubicin hydrochloride (Wako Pure Chem. Ind., Osaka, Japan), rhodamine 123 (R123; Sigma, St. Louis, MO, USA), verapamil (EGIS Hungarian Pharmaceutical Company, Budapest, Hungary), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma, St Louis, MO, USA), sodium dodecylsulfate (SDS; Sigma), dimethyl sulfoxide (DMSO; Sigma) and ethidium bromide (EB; Sigma, St. Louis, MO, USA). Stock solutions of R123 and verapamil were prepared in water. All solutions were prepared on day of assay.

Cell lines. L5178Y mouse T-cell lymphoma cells (ECACC cat. no. 87111908; U.S. FDA, Silver Spring, MD, USA) were transfected with pHa MDR1/A retrovirus, as described previously (12, 13). The ABCB1-expressing cell line was selected by culturing the infected cells with 60 ng/ml of colchicine (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) to maintain the MDR phenotype. L5178 mouse T-cell lymphoma cells (parental, PAR) and the human ABCB1-gene transfected sub-line (MDR) were cultured in McCoy's 5A medium supplemented with 10% heat-inactivated horse serum, L-glutamine (Lonza BioWhittaker, Verviers, Belgium) and antibiotics (penicillin, streptomycin) at 37°C and in a 5% CO₂ atmosphere.

Medium. McCoy's 5A medium (Lonza BioWhittaker) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich Química SA, Madrid, Spain), L-glutamine (Lonza BioWhittaker) and antibiotics.

Assay for antiproliferative and cytotoxic effect. The effects of increasing concentrations of the drugs alone on cell growth were tested in 96-well flat-bottomed microtitre plates. The compounds were diluted in a volume of 100 μl medium. Then, 6×10³ (for antiproliferative assay) or 2×10⁴ cells (for cytotoxic assay) in 50 μl of medium, respectively, were added to each well, with the exception of the medium control wells. The culture plates were further incubated at 37°C for 24 and 72 h, respectively; at the end of the incubation period, 15 μl of MTT (thiazolyl blue tetrazolium bromide, Sigma) solution (from a 5 mg/ml stock) was added to each well. After incubation at 37°C for 4 h, 100 μl of sodium dodecyl sulfate (SDS) (Sigma) solution (10% in 0.01 M HCI) was added to each well and the plates were further incubated at 37°C overnight. The cell growth was determined by measuring the optical density (OD) at 550 nm (ref. 630 nm) with Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA). Inhibition of the cell growth was determined according to the formula: Where ID₅₀ is defined as the inhibitory dose that reduces the growth of the compound-exposed cells by 50%.

Real-time fluorimetry was performed using a previously developed semi-automated method (14, 15). Briefly, the cells were adjusted to a density of 2×10⁶ cells/ml, centrifuged at 2000 × g for 2 min and re-suspended in phosphate-buffered saline (PBS) pH 7.4. The cell suspension was distributed in 90 μl aliquots into 0.2 ml tubes. The tested compounds were individually added at different concentrations (4 and 40 mg/l) in 5 μl volumes of their stock solutions and the samples incubated for 10 min at 25°C. Verapamil was used as a positive control. After this incubation, 5 μl (1 μg/ml final concentration) of EB (20 μg/ml stock solution) were added to the samples and the tubes were placed into a Rotor-Gene 3000™ thermocycler with real-time analysis software (Corbett Research, Sidney, Australia) and the fluorescence monitored on a real-time basis. The results were evaluated by Rotor-Gene Analysis Software 6.1 (Build 93) provided by Corbett Research.

Flow cytometry assay for evaluation of a compound on the retention of rhodamine 123 by MDR in tumour cells. This assay has been fully described previously (16). Briefly, the cells were adjusted to a density of 2×10⁶/ml, re-suspended in serum-free McCoy's 5A medium and distributed in 0.5 ml aliquots into Eppendorf centrifuge tubes. 10 μl of test compounds were added at various concentrations (4 and 40 mg/l), and the samples were incubated for 10 min at room temperature. Next, 10 μl (5.2 mM final concentration) of rhodamine 123 was added to the samples and the cells were incubated for a further 20 min at 37°C, washed twice and re-suspended in 0.5 ml phosphate-buffered saline (PBS) for analysis. The fluorescence uptake of the cell population was measured with FACStar Plus flow cytometer (Beckton, Dickinson and Company, Franklin Lakes, NJ, USA). Verapamil was used as a positive control in the rhodamine 123 exclusion experiments. The percentage mean fluorescence intensity was calculated for the treated MDR and parental cell lines as compared to untreated cells. A fluorescence activity ratio (FAR) was calculated via the following equation, on the basis of the measured fluorescence values: The results presented are obtained from a representative flow cytometry experiment in which 10,000 individual cells of the population were evaluated for amount of rhodamine 123 retained are first presented by the Beckton Dickinson FACStar flow cytometer as histograms and the data converted to FAR units that define fluorescence intensity, standard deviation, peak channel in the total and in the gated populations.

Checkerboard microplate method. The checkerboard microplate method was applied to study the drug interactions between resistance modifiers and anticancer drugs on cancer cells, as previously described (17). The interaction of the anticancer drug doxorubicin and the resistance modifiers hydantoins was studied in combination on MDR mouse T-lymphoma cells. The serial dilutions of doxorubicin (A) were made in 100 μl, horizontally, furthermore the dilutions of hydantoin derivatives (B) vertically, in 50 μl volume in the microtiter plate (the dilutions were prepared separately in Eppendorf tubes, using tissue culture medium). An aliquot of 50 μl of the cell suspension in tissue culture medium containing 2×10⁴ cells was distributed to each well and the plates incubated for 48 h at 37°C in a CO₂ incubator. The cell growth rate was determined after MTT staining and the intensity of the blue colour was measured with micro ELISA reader. Drug interactions were evaluated according to the following protocol, where ID₅₀ is defined as the inhibitory dose that reduces the growth of the compound-exposed cells by 50%.

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Figure 1.

Accumulation of EB (1 mg/l) by human MDR1 (ABCB1) gene-transfected mouse lymphoma cells in the presence of 4 and 40 μg/ml of AD-29.

• ID₅₀=50% inhibitory dose.

• FIC=fractional inhibitory concentration

• FIX=fractional inhibitory index

• FIC_A=ID_{50A in combination}/ID_{50A alone}

• FIC_B=ID_{50B in combination}/ID_{50B alone}