Evaluation of Cucurbitane-type Triterpenoids from Momordica balsamina on P-Glycoprotein (ABCB1) by Flow Cytometry and Real-time Fluorometry

Materials and Methods

Compounds tested. Four compounds with the cucurbitane skeleton, whose structures are presented in Figure 1, were tested for their anti-MDR activity: 7-methoxycucurbita-5,24-diene-3β,23(R)-diol (1), cucurbita-5,23(E)-diene-3β,7β,25,29-tetraol (2), 25-methoxycucurbita-5,23(E)-diene-3β,7β,29-triol (3) and 25-methoxycucurbita-5,23(E)-diene-3β-ol-7-O-β-D-allopyranoside (4). Compounds 1-4 were isolated and characterized previously by Ramalhete et al. (13) from the methanol extract of Momordica balsamina. All the compounds were dissolved in DMSO. The plant was collected in Gaza, Mozambique (August 2006). The plant was identified by Dr. Silva Mulhovo, and a voucher specimen (30 SM) has been deposited at the herbarium (LMA) of the Instituto de Investigação Agronómica of Mozambique.

Cell lines. L5178 mouse T-cell lymphoma cells were transfected with pHa MDR1/A retrovirus, as described elsewhere (11, 12). The ABCB1-expressing cell lines were selected by culturing the infected cells with 60 ng/ml of colchicine 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 (Lonza BioWhittaker, Verviers, Belgium) supplemented with 10% heat-inactivated horse serum (Sigma-Aldrich Química SA, Madrid, Spain), L-glutamine (Lonza BioWhittaker) and antibiotics (penicillin, streptomycin) at 37°C and in a 5% CO₂ atmosphere.

Assay for antiproliferative 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 50 μl medium and 1×10⁴ cells in 0.1 ml of medium were added to each well, with the exception of the medium control wells. The culture plates were incubated at 37°C for 72 h; at the end of the incubation period, 15 μl of dimethyl thiazolyl blue tetrazolium bromide (MTT; Sigma, St. Louis, MO, USA) solution (from a 5 mg/ml stock) were added to each well. After incubation at 37°C for 4 h, 100 μl of sodium dodecyl sulfate (SDS) (Sigma) solution (10%) were measured into 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 a 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%. The ID₅₀ values are expressed as means±SD from three experiments.

EB accumulation assay. The cells were adjusted to a density of 2×10⁶ cells/ml, centrifuged at 2,000×g for 2 minutes and re-suspended in phosphate-buffered saline (PBS) at 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 (3 and 30 μM) in 5 μl volumes of their stock solutions and the samples were then incubated for 10 minutes at 25°C. Verapamil was used as a positive control (10). 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. Prior to the assay, the instrument was programmed for temperature (37°C), the appropriate excitation and emission wavelengths of EB (530 nm bandpass and 585 nm highpass, respectively), and the time and number of cycles for the recording of the fluorescence (9). The results were evaluated by Rotor-Gene Analysis Software 6.1 (Build 93) provided by Corbett Research. From the real-time data, the relative final fluorescence (RFF) of the last time point (minute 60) of the EB accumulation assay was calculated according to the formula given in Table III. The RFF value is the difference between the relative fluorescence (RF) at the last time point of the EB retention curve of cells in the presence of an inhibitor and the RF at the last time point of the EB retention curve of the untreated solvent control.

Flow cytometric assay for evaluation of a compound based on the retention of rhodamine 123. 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. Test compounds were added at different concentrations in (10 μl of 0.025-1 mM), and the samples were incubated for 10 min at room temperature. Subsequently, 10 μl (5.2 mM final concentration) of the indicator rhodamine 123 were 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 PBS for analysis. The fluorescence uptake of the cell population was measured with FACS Star Plus flow cytometer (Beckton, Dickinson and Company, Franklin Lakes, NJ, USA). Verapamil was used as a positive control. The percentage mean fluorescence intensity was calculated for the treated MDR and PAR 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 were obtained from a representative flow cytometric experiment in which 10,000 individual cells of the population were evaluated for the amount of rhodamine 123 retained.