Modulation of Multidrug Efflux Pump Activity by New Hydantoin Derivatives on Colon Adenocarcinoma Cells without Inducing Apoptosis

Materials and Methods

Compounds. Eight hydantoin derivatives (SZ-7, BS-1, JH-63, MN-3, RW-15b, AD-26, RW-13, AD-29) were tested, kindly provided by Dr. Jadwiga Handzlik and Prof. Dr. Katarzyna Kiec-Kononowicz, Cracow, Poland. The compounds were dissolved in DMSO.

Cell lines. The human colon adenocarcinoma cell lines (Colo 205 doxorubicin sensitive parent and Colo 320/MDR-LRP resistant to anticancer agents overexpressing ABCB1(MDR1)-LRP), ATCCCCL-220.1 (Colo 320) and CCL-222 (Colo 205), were purchased from LGC Promochem, Teddington, England.

The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 1 mM Na-pyruvate and 100 mM Hepes. The cells were incubated in a humidified atmosphere (5% CO₂, 95% air) at 37°C. The semi-adherent cancer cells were detached with 0.25% trypsin and 0.02% EDTA for 5 min at 37°C.

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-Aldrich Chemie GmbH, Steinheim) 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 HCl) was added to each well and the plates were further incubated at 37°C overnight. Cell growth was determined by measuring the optical density (OD) at 550 nm (ref. 630 nm) with a Multiscan EX ELISA reader (Thermo Labsystems, Cheshire, WA, USA).

Inhibition of the cell growth was determined according to the formula: Where IC₅₀ is defined as the inhibitory dose that reduces the growth of the compound-exposed cells by 50%.

Flow cytometry assay. Functional analysis of the ABCB1 transporter can be accomplished using flow cytometric accumulation assays with fluorescent dyes (9); for this purpose the intracellular accumulation of the non-toxic fluorescent ABCB1 (P-glycoprotein) substrate rhodamine 123 by the Colo 320 cells with an overexpressed ABCB1 efflux system was studied. This assay has been fully described previously (10). Briefly, the cells were adjusted to a density of 2×10⁶/ml, re-suspended in serum-free RPMI 1640 medium and distributed in 0.5 ml aliquots into Eppendorf centrifuge tubes. The test compounds (10 μl) were added at various concentrations 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 a 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 Colo 320 and sensitive Colo 205 cells 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 obtained from a representative flow cytometry experiment in which 10,000 individual cells of the population were evaluated for the quantity of rhodamine 123 retained with the aid of the Beckton Dickinson FACStar flow cytometer, were first presented as histograms and the data was converted to FAR units that define fluorescence intensity, standard deviation, peak channel in the total- and in the gated- populations.

Assay for apoptosis induction. The assay was carried out using an Annexin V-FITC Apoptosis Detection Kit Cat. No. PF 032 from Calbiochem (EMD Biosciences, Inc. La Jolla, CA) according to the manufacturer's instructions.

The concentration of the cell suspension was adjusted to approximately 1×10⁶ cells/ml. The cell suspension was distributed into 0.5 ml aliquots (5×10⁵ cells) into a 24-well microplate and incubated overnight, at 37°C, 5% CO₂. On the following day the medium was removed and fresh medium was added to the cells. The cells were incubated in the presence of the hydantoin derivatives for 3 hours at 37°C; the concentration for the apoptosis induction (4 or 40 mg/l) were selected based on previous results (8).12H-benzo[α]phenothiazine was used as positive control at a final concentration of 50 μg/ml. The culture medium was removed, the cells were washed with PBS and fresh medium was added to the cells. The 24-well plates were incubated overnight at 37°C, 5% CO₂.

After the incubation the supernatant was collected in a microfuge tube and 200 μl of 0.25 trypsin (Trypsin-Versen) was added to the wells until the cells detached from the surfaces of the wells. After the addition of culture medium, the cells were centrifuged at 2000 × g for 2 min at room temperature, the supernatant was removed and the cells were re-suspended in fresh serum-free medium.

After this procedure, the apoptosis assay was carried out according to the rapid protocol of the kit. The other conditions including AnnexinV-FITC and propidium iodide controls were applied as described elsewhere (11). The fluorescence was analysed immediately using a Becton Dickinson FACStar flow cytometer.