Effects of Structural Modifications of Daunorubicin on In Vitro Antileukemic Activity

Materials and Methods

Cells. Human acute lymphoblastic leukemia MOLT-4 cells and human acute myeloblastic leukemia ML-1 cells (European Collection of Cell Cultures, Salisbury, Wiltshire, UK) were maintained in RPMI-1640 medium (Gibco BRL Life Technologies, Warsaw, Poland) supplemented with 10% fetal calf serum (Gibco BRL Life Technologies, Warsaw, Poland), 2 mM L-glutamine (Sigma Aldrich, Poznań, Poland), and antibiotic antimycotic solution (AAS; Sigma Aldrich, Poznań, Poland). AAS contained 20 units of penicillin, 20 μg streptomycin and 0.05 μg amphotericin B. Every third day, the acute leukemia cells were passaged. The cells grew at 37°C in an atmosphere of 5% CO₂ in air (HERAcell incubator; KendroLab, Warsaw, Poland).

Chemicals. The three anthracycline compounds (Figure 1), daunorubicin and two formamidinodaunorubicins DAUFmor and DAUFhex were synthesized at the Institute of Biotechnology and Antibiotics (Warsaw, Poland). The formamidinodaunorubicin compounds were obtained in the reaction of daunorubicin hydrochloride with acetals, derivatives of N-formylamines. The structure of the formamidinodaunorubicins were confirmed by their ¹H-nuclear magnetic resonance (NMR) and ¹³C-NMR spectra. According to the high preasure liquid chromatography (HPLC) analysis, the purities of DAUFmor and DAUFhex were 98.8 and 99.2%, respectively (4). Daunorubicin, DAUFmor, and DAUFhex were dissolved in aqua pro injectione (Polpharma, Starogard Gdański, Poland). All solutions were freshly-prepared directly before treatment of the acute leukemia cells.

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Figure 1.

Chemical structures of daunorubicin (DAU), DAUFmor, and DAUFhex.

Anthracycline doses and cell treatment. After a dilution of the cell suspension to a density of 15×10⁴ cells/ml medium, MOLT-4 and ML-1 cells were subjected to the anthracycline agent exposure. MOLT-4 cells were exposed to DAU, DAUFmor, and DAUFhex at concentrations of 25 nM, 50 nM, and 75 nM, respectively, and ML-1 cells to the action of these anthracycline agents applied at the concentrations of 75 nM, 150 nM, and 225 nM, respectively. The controls consisted of untreated MOLT-4 and ML-1 cells. The chosen concentrations of daunorubicin, DAUFmor, and DAUFhex used in the present studies are based on the unpublished data of our previous experiments performed on these two human acute leukemia cell lines.

Analyses of leukemia cells after the anthracycline application. The temporary changes occurring in human acute lymphoblastic leukemia MOLT-4 cells and human acute myeloblastic leukemia ML-1 cells were observed at 24 h, 48 h, and 72 h after exposure to daunorubicin and its two formamidinodaunorubicin derivatives. At these three time intervals, the cell viability, volume and count were assessed.

The assays were conducted using the spectrophotometric 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and the electronic Beckman-Coulter method. In viable, metabolically active cells, the tetrazolium ring of MTT is cleaved, yielding formazan crystals. Changes in the metabolic activity of cell populations result in a concomitant change in the amount of formazan formed. The Beckman-Coulter method of cell sizing and counting is based on the detection of an electrical pulse which results from the passage of each cell through an aperture. The amplitude of the produced electrical pulse depends on the cell volume. The number of pulses indicates the cell count.

In vitro spectrophotometric MTT assay: MTT (Sigma-Aldrich) was dissolved in RPMI 1640 medium, at a concentration of 5 mg/ml, and filtered through a 0.2-μm filter. Subsequently, 100 μl of the yellow MTT solution was added to each well of a 24-well plate, containing 1 ml of the cell suspension. The cells were then incubated at 37°C with 5% CO₂. A blank solution was prepared according to the above procedure using complete medium without cells. After a three-hour incubation period, the resulting formazan crystals were dissolved with 1 ml of acidified isopropanol (0.05 N HCl in absolute isopropanol), and the absorbance of the obtained solution was measured at wavelength of 570 nm using a Pharmacia Ultrospec III spectrophotometer (Pharmacia LKB Biotechnology, Uppsala, Sweden).

The half maximal inhibitory concentration (IC₅₀) value determination: The half maximal inhibitory concentration is a measure of the effectiveness of a compound in inhibiting biochemical processes and biological functions. According to the in vitro MTT assay, the IC₅₀ represents the concentration of the tested anthracycline agent that is required for 50% inhibition of the human leukemia cell viability. Based on the obtained data using the in vitro MTT assay, the IC₅₀ values for daunorubicin and its two analogs, DAUFmor and DAUFhex, calculated separately, at 72 h after the human leukemia cells exposure to the anthracycline agents. To determine the IC₅₀ values, the concentration range used of each antracycline was from 10-1,000 nM.

Measurement of cell size and count: Samples of the acute leukemia cell suspension (500 μl) were taken from flasks and immediately diluted in 4.5 ml ISOTON II (Beckman-Coulter filtered electrolyte solution based on 0.9% saline). After the dilution of the leukemia cell suspension, individual cells were measured using a Z2 Coulter counter (Beckman-Coulter, Miami, FL, USA). The volume and count distribution of leukemia cells was obtained using a counter equipped with a 100 μm diameter orifice. The flow rate was 500 μl/12.5 s. The range for MOLT-4 cell measurement was determined as 65-3674 fL and for ML-1 cell measurement as 180-8181 fL. The volume and count of MOLT-4 cells and ML-1 cells were analyzed, respectively, at 402-3674 fL, and at 459-7346 fL. The instrument was calibrated using 10 μm diameter latex beads (Beckman-Coulter CC size standard). The mean cell volume and the cell count were determined using Z2 AccuComp software (Beckman-Coulter, Miami, FL, USA).

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Figure 2.

Effects of daunorubicin (DAU), DAUFmor, and DAUFhex on the viability of MOLT-4 cells (A) and ML-1 cells (B). The extent of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) conversion to formazan in the acute leukemia cells reflects their viability. Values not significantly different at p<0.05 according to the Duncan's test: *, **, *** between the groups of leukemia cells treated with the anthracycline agents; #compared to control; +between the time points.

Statistical evaluation. Statistical significance of differences in the amount of formazan formed, the IC₅₀ value, and the cell volume and count were evaluated by an analysis of variance and the Duncan's new multiple range test. A difference with p<0.05 was considered statistically significant. The results were confirmed by three independent experiments carried out in triplicate.