Chemotherapeutic Resistance, Cytotoxicity, and Inhibitor Efficacy Assays

Drug resistance was quantified by comparing the percent confluency of Igrov1/T8 cells and Igrov1 parental cells maintained in 950 nM TPT and the effective concentration of topotecan (TPT) that resulted in 50% cell death (EC₅₀) of Igrov1 parental and Igrov1/T8 cells. To determine that Igrov1/T8 cells were resistant to TPT, Igrov1 parental and T8 cells were plated at 50% confluency on day 0 in a concentration of 950 nM TPT. Percent confluency was estimated under light microscopy everyday for 14 days. Once the Igrov1/T8 cells grew to 100% confluency (day 7) they were split down to 50% confluency on that day to prevent overgrowth and cell death. A conversion factor of 2 was used for Igrov1/T8 confluency on days 7 to 14 for an estimate of continued cell growth. To determine the EC₅₀ of resistant and parental cells, Igrov1 Parental and ABCG2-overexpressing Igrov1/T8 cells were separately placed into 6-well plates such that the total number of cells in each well was approximately 1.5 × 10⁵ in a total volume of 2 ml (7.5 × 10⁴ cells/ml). TPT was added to the Igrov1 parental and Igrov1/T8 wells at increasing concentrations from 0 to 3 μM and cell viability was determined on days 3 and 7. Similar experiments were used to evaluate the cytotoxicity and efficacy of each ABCG2-selective inhibitor by examining the median lethal dose (TD₅₀) or the half-maximal inhibitory concentration (IC₅₀) of each compound.(28)