Cell viability

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted to test the effect of probucol on the various human cancer cell lines and mouse fibroblast cells. For this purpose, cells (20,000 cells/well) were seeded onto 96-well plates. 20 mM stock solution of probucol was pepared by dissolving probucol in dimethyl sulfoxide (DMSO) and further dilutions were prepared with culture medium. Maximum concentration of probucol applied to cells was 10 µM, due to low solubility of the compound at higher concentrations. H929, U266, RPMI8226, U937, and HL60 cells were treated with 0.1-10 µM probucol for 72 h at 37°C. Control cells were incubated with the same concentration of DMSO as the probucol-treated cells and the DMSO concentration never exceeded 0.5%. For each cell line, the same protocol was followed. K562S (imatinib sensitive) and K562 R (imatinib resistant) cells were treated with probucol 0.1-10 µM), imatinib, and imatinib/probucol combination for 72 h. Imatinib concentrations used for K562S and K562R cells were 0.5 µM and 20 µM, respectively. Since imatinib is a first line therapeutic option for CML, we determined its single effect or combination effect with probucol. The effect of probucol was also detemined in non-cancerous L929 fibroblast cell line. After proper incubation, MTT solution (5 mg/mL) was prepared with phosphate buffer saline and cells were incubated with MTT solution for a period of four hours. Insoluble formazan crystals were dissolved by SDS-HCl solution. Absorbance was measured at 550 nm using a microplate reader (Molecular Devices-Spectra Max spectrophotometer, Sunnyvale, CA, USA).