Cytotoxicity assay

The in vitro cytotoxicity of DOX, OXP and stattic was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. B16F10 and CT26 cells (0.3 × 10⁴) were seeded into 96-well plates and grown overnight. Then, the cells were treated with each compound or combination of two compounds at different concentrations. Untreated and dimethyl sulfoxide (DMSO)-treated cells were considered as control cells. After 48 h incubation, the cells were added with MTT solution (0.5 mg/mL) and the cells were incubated for further 4 h at 37 °C. Finally, the medium was removed, DMSO was added and the absorbance was read at 570 nm by a microplate reader (BioTek Instruments, Inc. USA). Dose-response curve and IC₅₀ value (the half-maximal inhibitory concentration) for either agent were calculated by Graphpad prism software. The combination index (CI) values for each dose were determined using data obtained from MTT assays and CompuSyn software. According to Chou-Talalay method, quantitative definition CI > 1.1, CI = 0.9–1.1 and CI < 0.9 have respectively antagonistic, additive and synergistic effect in drug combination [26]. The concentrations of drugs for combination therapy experiments were selected based on IC₅₀ value of each compound in the selected cell lines. We choose to use the drugs at the concentration lower than their IC₅₀ to avoid losing a major part of the cells due to the cytotoxicity of the compounds at higher concentrations. Besides, combination therapy with two drugs at the selected concentrations was found to result in an optimal anticancer effect indicated with CI.