Evaluating Drug Resistance in Colo 320 Cells

In order to test the viability of drug-resistant Colo 320 cells before and after treatments with chemotherapy drugs or synthetic steroids, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell viability assays were conducted (first described by Mosmann [42 (link)]). First the toxicity of chemotherapy drugs was determined. Colo 320 cells were seeded in 96-well plates at 10⁴ cells/well density and were left to grow for 24 h. To obtain dose-response curves, cells were treated with a serial dilution of each drug (Bleomycin, Carmustine, Cisplatin, Doxorubicin, Epirubicin; treatment was applied in 200 μL media) for another 24 h (for the applied concentrations, please refer to Supplementary Material Table S1). Then, the media were discarded and replaced with 100 μL fresh media containing 0.5 mg/mL MTT reagent (Sigma-Aldrich, St. Louis, MO, USA). After a 1 h incubation, this media was replaced by 100 μL DMSO to solubilize formazan crystals. Absorbance of the samples was measured at 570 and 630 nm with a Synergy HTX microplate reader (BIOTEK^®, Santa Clara, CA, USA). Untreated cells were considered as 100% cell viability during data analysis. IC₅₀ and the Hill-slope values were obtained from the measured data. The IC₁₀–IC₉₀ values for each drug were calculated from the IC₅₀ and the Hill-slope values.
We tested also the toxicity of androstano-arylpyrimidine 17-acetates. For this, drug-sensitive Colo 205 and multidrug-resistant Colo 320 cells were exposed to compounds 10a, 10d, 10e, 10f, and 10g in a fixed 20 μM concentration for 24 h, then MTT viability assay was performed. Cell seeding and absorbance measurement were carried out in the same way as described above.
For the combinational treatments (chemotherapy drug + steroid), each chemotherapy drug was applied in its previously determined respective IC₃₀–IC₇₀ concentration (in the case of each drug, the appropriate concentrations are presented in Supplementary Material Table S2, indicated in the first column of the table next to the drug name) together with each androstano-arylpyrimidine 17-acetate (10a, 10d, 10e, 10f, and 10g). The steroids were used in a fixed 20 μM concentration. Based on these treatments, certain combinations were selected to be utilized later for an apoptosis detection assay.