[3H]-paclitaxel accumulation and efflux

Intracellular paclitaxel accumulation and efflux were measured in HEK/pcDNA cells and HEK/MRP7 cells. For 4 h accumulation assay, the cells were trypsinized and three aliquots (5×10⁶ cells) from each cell line were resuspended in the medium. To measure drug accumulation, cells were preincubated in DMEM in the presence or absence of tariquidar (at 0.1 and 0.3 µM) or cepharanthine (at 2.5 µM) for 2 h, washed and then incubated with 0.01 µM [³H]-paclitaxel with or without tariquidar (at 0.1 µM and 0.3 µM) or cepharanthine (at 2.5 µM) for another 2 h at 37°C. The cells were pelleted at 4°C, washed twice with 10 ml ice-cold PBS, and lysed in 1% SDS. Radioactivity was measured in a liquid scintillation counter. For 72 h accumulation assay, cells were cultured in DMEM in the absence or presence of tariquidar for 70 h. Cells were then trypsinized and resuspended with the same cell number in each group, and incubated with 0.01 µM [³H]-paclitaxel with or without tariquidar (at 0.3 µM) for another 2 h at 37°C. After this, the steps were the same as 4 h accumulation. For the efflux study, cells were incubated with 0.01 µM [³H]-paclitaxel according to the method for the accumulation study. After being washed twice with cold PBS, the cells were cultured in fresh DMEM with or without 0.3 µM tariquidar at 37°C. After 0, 30, 60 or 120 min, aliquots of cells were removed and immediately washed with ice-cold PBS. The cell pellets were collected for radioactivity measurement in a Packard TRI-CARB® 1900CA liquid scintillation analyzer (Packard Instrument Company, Downers Grove, IL).