Cytotoxicity assay and the calculation of resistant fold and selectivity index

The cytotoxicity of the hormone steroids was evaluated by the sulforhodamine B (SRB) assay through determination of total protein amounts. Briefly, after 72 h treatment with drugs, 50% trichloroacetic acid (TCA) was added for 30 min to fix cells, which then were washed with water and air-dried. Afterward, cells were stained with 0.04% SRB for 30 min and then washed with 1% acetic acid to remove unbound dye and air-dried. The bound stain was solubilized in 10 mM Tris Base and the absorbance was measured on a BioTek Synergy HT Multi-Mode Microplate Reader at 515 nm. Three dose response parameters were calculated for each experimental agent. Growth inhibition of 50% (IC₅₀) was calculated from [(Ti − Tz)/(C − Tz)] × 100 = 50 (Tz: time zero; C: control growth; Ti: growth of tested group in the presence of drug at the different concentration levels), which is the drug concentration resulting in a 50% reduction of the net protein increase (as measured by SRB staining) in control cells during the drug incubation.

The Resistance Fold (RF) was calculated by dividing the individual IC₅₀ of MDR cell line (KB/VIN, HepG2/VIN, or NCI-H460/MX20) by the IC₅₀ of parental cell line (HeLaS3, HepG2, or NCI-H460). The Selectivity Index (SI) was calculated by dividing the individual IC₅₀ of parental cell line (HeLaS3, HepG2, or NCI-H460) by the IC₅₀ of MDR cell line (KB/VIN, HepG2/VIN, or NCI-H460/MX20). A higher RF value indicates that the MDR cancer cells showed higher resistance to the compound, while a higher SI value indicates that the MDR cancer cells showed higher response to the compound.