In vitro cytotoxicity assay

Human cancer cell lines, i.e., HepG2, A498, NCI-H226, H157 and MDR-2780AD were purchased from American Type Culture Collection (ATCC). The cells were grown and maintained in RPMI 1640 medium supplemented with foetal bovine serum at 37 °C, 5% CO₂ and 90% humidity.

The assay was performed in 96-well microtitre plates. The cells were seeded separately to 96-wells plates at a concentration of 1 × 10⁵ cells/well. These cells were then treated with different concentrations of compound 1 (5–100 μM) with three replicates of each concentration. The control contained only the cells without test sample. The culture plates were then incubated at 37 °C for 72 h in a humidified incubator. After 72 h of incubation, the fraction of surviving cells was measured relative to the cell population in the control by colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Mossmann 1983). A volume of 20 μL of MTT (5 mg/mL) in phosphate buffer solution was added to each well and incubated for 3–4 h. After this incubation, 100 μL of DMSO was added to dissolve the resulting MTT formazan crystals by pipetting up and down 10–20 times. The plates were left at room temperature for 15–30 min then the optical density (OD) was measured on an ELISA microplate reader at 570 nm. The percentage of cell viability was calculated using the following equation:

A plot of cell viability against the concentration of the sample gave a measure of cytotoxicity. The IC₅₀ was then calculated from the plot.