Cabazitaxel was purchased from MedChemexpress (Manmouth Junction, NJ, USA). Paclitaxel was purchased from Tocris Bioscience (Ellisville, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), 10,000 IU/mL penicillin and 10,000 μg/mL streptomycin, and 0.25% trypsin were purchased from HyClone (Waltham, MA, USA). 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), ammonium molybdate, 2-(N-morpholino) ethanesulfonic acid (MES) hydrate, antimony potassium tartrate, sodium azide, and N-methyl-D-glucamine were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Potassium phosphate, ethylene glycol tetraacetic acid (EGTA), and adenosine triphosphate (ATP) were products of AMRESCO (Solon, OH, USA). Sulfuric acid solution (37 N) was purchased from Fisher Scientific (Pittsburgh, PA, USA). KCl was purchased from Avantor Performance Materials (Center Valley, PA, USA). Ouabain was purchased from Enzo Life Sciences, Inc. (Farmingdale, NY, USA). Dithiothreitol was purchased from Promega Corporation (Madison, WI, USA). MgCl2 was purchased from EMD Millipore (Billerica, MA, USA). Ascorbic acid was purchased from VWR International (West Chester, PA, USA). Sodium orthovanadate was purchased from Alfa Aesar (Ward Hill, MA, USA). The OPSYS microplate reader was purchased from Dynex Technologies (Chantilly, VA, USA).
The ABCB1-overexpressing KB-C2 cell line was established by a step-wise exposure of KB-3-1 cells, a parental human epidermoid carcinoma cell line, to increasing concentrations (up to 2 μg/mL) of colchicine . The LLC-PK1 porcine renal epithelial cells were transfected with wild-type human ABCB1 cDNA plasmid as previously described, and the stable transfectant cell line was named LLC-MDR1-WT . HEK293/pcDNA3.1 and HEK293/ABCC10 cells were generated by transfecting HEK293 cells with an empty vector and an ABCC10 expression vector, respectively . We thank Dr. Shinichi Akiyama (Kagoshima University, Japan) for the KB-3-1 and KB-C2 cell lines, Dr. Michael M. Gottesman (NCI, NIH, USA) for the LLC-PK1 and LLC-MDR1-WT cell lines, and Dr. Gary D. Kruh (University of Illinois at Chicago, IL, USA) for the ABCC10 plasmid.
To determine the drug sensitivities of the previously described ABCB1-overexpressing KB-C2 cells, LLC-MDR1-WT cells, and ABCC10-overexpressing HEK293/ABCC10 cells, with KB-3-1, LLC-PK1, and HEK293/pcDNA3.1 cells as the respective controls [1,16], a modified MTT assay was performed [1,21]. Approximately 4,000 KB-3-1 cells, 7,000 KB-C2 cells, and 5,000 LLC-PK1, LLC-MDR1-WT, HEK293/pcDNA3.1, and HEK293/ABCC10 cells were seeded in 180 μL of medium in each well of 96-well plates. After incubating for 24 h at 37°C, 20 μL of paclitaxel or cabazitaxel (0.01 to 10 μmol/L) was added. Subsequently, cells treated with paclitaxel or cabazitaxel in DMEM supplemented with 10% FBS were incubated at 37°C for 72 h. After 72 h, 20 μL MTT (4 mg/mL) was added to each well. The cells were incubated at 37°C for another 4 h. The MTT with medium was removed, and 100 μL of DMSO was added to each well. The absorbance was measured at 570 nm by an Opsys microplate reader (Dynex Technologies, VA, USA). The degree of resistance was calculated by dividing the 50% inhibition concentration (IC50) as calculated using the Bliss method for drug-resistant cells (KB-C2, LLC-MDR1-WT, and HEK293/ABCC10) by that of the parental drug-sensitive cells (KB-3-1, LLC-PK1, and HEK293/pcDNA3.1), respectively. Each MTT assay was run in triplicate.
ABCB1 ATPase assay
The ABCB1 transporter uses energy derived from the hydrolysis of ATP to efflux their substrates across the membrane against a concentration gradient; thus, the ATP consumption reflects the ATPase activity of the transporter. The vanadate (Vi)-sensitive ATPase activity of ABCB1 in the membrane vesicles of High Five insect cells [(His)-6-tagged ABCB1 expressed in Trichoplusia ni cells using the recombinant baculovirus system and purified by metal affinity chromatography] was measured as previously described [22,23]. The membrane vesicles (10–20 μg protein/reaction) were incubated in ATPase assay buffer (50 mmol/L MES-Tris, pH 6.8, 50 mmol/L KCl, 5 mmol/L sodium azide, 1 mmol/L EGTA, 1 mmol/L ouabain, 2 mmol/L dithiothreitol, and 10 mmol/L MgCl2) at 37°C for 5 min with or without 0.3 mmol/L vanadate. The membrane vesicles in ATPase assay buffer were incubated with different concentrations (0–10 μmol/L) of paclitaxel or cabazitaxel at 37°C for 3 min, and then 5 mmol/L ATP was added at 37°C. After 20 min of incubation, the reaction was terminated by adding 0.1 mL of 5% SDS solution. The amount of Pi released was quantified at 800 nm using a Bio-Rad SmartSpec Plus Spectrophotometer (Hercules, CA, USA) as previously described [23,24].
All experiments were repeated at least three times. The differences between runs were assessed using the two-tailed Student’s t-test, and statistical significance was determined at P < 0.05. Microsoft Office Excel 2010, licensed from Microsoft (Redmond, WA, USA), was used for data processing and analysis.