Cytotoxicity of Endogenous Lipids N-acyl Dopamines and their Possible Metabolic Derivatives for Human Cancer Cell Lines of Different Histological Origin

Materials and Methods

Human neuroblastoma IMR-32 and osteosarcoma HOS cell lines were purchased from the cell bank of the Russian Collection of Cell Cultures (Institute of Cytology, Russian Academy of Sciences, Saint-Petersburg, Russia). Human lymphoma K-562, leukemia Namalwa, carcinoma MCF-7 and HEK-293 cell lines were a kind gift of Dr. E. Smirnova (IBCh RAS, Russia). DMEM, RPMI 1640 and MEM cell medium, Hank's solution, HEPES, phosphate-buffered saline, streptomycin, penicillin, amphotericin B, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), non-essential amino acids solution and trypsin-EDTA solution were from PanEco, Moscow, Russia. D-glucose, diaphorase, NAD⁺, DL-lactic acid, Tris, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT), bovine serum albumin, sucrose and sodium pyruvate were from Sigma-Aldrich, St. Louis, MO, USA. Fetal bovine serum was from PAA Laboratories, Pasching, Austria.

All test compounds were synthesized in the Laboratory of Oxylipins of Shemyakin-Ovchinnikov Institure of Bioorganic Chemistry, RAS, Russia as previously described (1) and stored only as ethanol or dimethyl sulfoxyde (DMSO) stocks under argon atmosphere at −52°C.

All cell lines were maintained at 37°C under 5% CO₂ in the medium (see below), supplemented with 2 mM L-glutamine, 25 mM HEPES (9), 10% fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin and 0.25 μg/ml amphotericin B. MCF-7 and HEK-293 were cultured in DMEM medium, IMR-32 in Eagle's MEM medium supplemented with 1 mM sodium pyruvate, while Namalwa and K-562 were grown in RPMI 1640 medium. The medium for MCF-7 and HEK-293 cells was additionally supplemented with 1% non-essential amino acids solution. Cells were passaged each 3 days and continiously grown for no more than 40 passages. Attached cells were detached with 0.25% trypsin in 0.53 mM EDTA in Hanks' salts. Cells were counted using a glass hemocytometer.

For analysis of toxicity of NADA and related compounds, cells were plated in 96-well plates at a density of 1.25×10⁴ cells per well and grown for 3 days. Serial dilutions of test compounds (the range of final concentrations after addition to cells of 0.1-100 μM) prepared in DMSO and dissolved in the culture medium were added to the cells in triplicate for each concentration and incubated for 18 h. The incubation time was chosen on the basis of the most pronounced differences between the compounds tested. The final DMSO concentration was 0.5%. Negative control cells were treated with 0.5% DMSO. Positive control cells were treated with 3.6 μl of 50% Triton X-100 in ethanol per 200 μl of cell culture medium. Separate controls were without DMSO (no difference with the control 0.5% DMSO was found, data not shown). The effect of test substances on cell viability was evaluated using both MTT test (based on the MTT dye reduction by mitochondria of living cells) (13) and lactate dehydrogenase (LDH) test (based on the lactate dehydrogenase liberation from the dead cells) (14).

For the MTT test, after removal of the medium with the test compounds, cells and controls were incubated for 1.5 h with 0.5 mg/ml of MTT in Hank's solution, supplemented with 10 mM of D-glucose. After this incubation, the solution was removed and the cells were dissolved in DMSO. For the suspension cells, 10× stock of MTT dye in Hank's solution was added directly to the medium with the test compounds; after the incubation, an equal volume of 0.1 N HCl in isopropanol was added to the medium to dissolve the formazan crystals formed by the cells from the MTT. In this case, we also used a separate control with substances in the medium without cells to compensate for the interaction of NADA with the MTT dye. The amount of the reduced dye was determined colorimetrically at 594 nm with a reference wavelength 620 nm using an EFOS 9505 photometer (Sapphire, Moscow, Russia). Additionally, the attachment and cell shape of adherent cells were evaluated microscopically.

For the analysis of the LDH activity, to a 60 μl aliquot of the cell medium 1 μl of 50% Triton X-100 in ethanol, 8 μl of 36 mg/ml DL-lactate in Tris-HCl pH 8.5, 8 μl of 2 mg/ml INT and 8 μl of 3 mg/ml NAD⁺ mixed with 13.5 U/ml diaphorase were sequentially added. The last two compounds were dissolved in phosphate-buffered saline with 1.2% sucrose and 0.03% bovine serum albumin prior to each experiment. INT stock solution (10×) was prepared in DMSO and stored at −20°C. The reaction mixture was incubated for 20 min at room temperature after which the reaction was terminated with 5 μl of 3N HCl. Its optical density was determined at 490 nm using an EFOS 9505 photometer (Sapphire). Cell medium with purified lactate dehydrogenase (0.05 U/ml) was incubated with the test substances as a separate control; neither one inhibited the enzyme in the concentration range used.

Each experiment was repeated three times. Curves for calculating the 50% lethal dose (LD₅₀) were generated using the GraphPad Prism software (www.graphpad.com); the substances that caused ≤10% cell death at 100 μM were considered inactive. Data are presented as mean±standard deviation. Data were compared using the unpaired Student's t test; p values of 0.05 were considered significant.