PXR-mediated induction of P-glycoprotein by anticancer drugs in a human colon adenocarcinoma-derived cell line

The pGL3-MDR1 (p-10224) luciferase reporter construct was generously provided by Dr. Oliver Burk (Institute of Clinical Pharmacology, Eberhard-Karls University, Tübingen, Germany). The pCDG-hPXR expression vector was generously provided by Dr. Ron Evans (Salk institute for biological studies, La Jolla, CA, USA). The pRL-TK control plasmid was obtained from Promega (Madison, WI, USA). Plasmids were checked by enzyme restriction and agarose gel electrophoresis and purified using Promega’s Pureyield Midi-prep (Madison, WI, USA) according to the instructions of the manufacturer.

The human colon adenocarcinoma-derived cell line LS180 was purchased from the ATCC (Manassas, VA, USA). The cell-line was maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (with 25 mM HEPES and l-glutamine, supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin), at 37°C under a humidified atmosphere of 5% CO₂.

Cell viability was after 48 h, and the anticancer drug treatment was assessed with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-assay.

The Pgp reporter gene assay was performed in a similar manner as described previously [10]. In brief, LS180 cells were transfected with a pCDG-hPXR expression vector, a pGL3-MDR1 luciferase reporter construct, and a pRL-TK control plasmid for 24 h. After transfection, the transiently transfected LS180 cells were treated with the highest non-toxic concentration of each anticancer drug. After 48 h incubation, the reporter activity was determined.

LS180 cells were plated at a density of 5 × 10⁴ cells/well in 96 well plates (Pgp protein level determination) or 1 × 10⁵ cells/well in 1 ml RMPI in 24 well plates (Pgp transport activity). After reaching 80–90% confluency, medium was replaced with medium containing the different anticancer drugs: carboplatin (10 μM), cyclophosphamide (100 μM), ifosfamide (100 μM), docetaxel (10 μM), paclitaxel (10 μM), flutamide (10 μM) and tamoxifen (10 μM). Rifampicin (10 μM) was used as a positive control and DMSO (0.1%) as a negative control. The cells were treated for 48 h with the drugs and the controls. At the end of each treatment, the cells were processed for immunoblotting or rhodamine 123-based Pgp activity assays.

The siRNA sequence targeting human PXR (sense: cguuuguucgcuuccugagtt; antisense: cucaggaagcgaacaaacgtg) and the negative control, which consisted of a non-complementary sequence, were purchased from Ambion (Austin, TX, USA). LS180 cells were reversely transfected 48 h prior to the Pgp induction experiment with 50 nM siRNA PXR or 50 nM negative control siRNA using Lipofectamine RNAi Max (Invitrogen, Breda, The Netherlands). Subsequently, PXR knockdown and control LS180 cells were treated with different anticancer drugs for 48 h.

After 48 h of culture, cells were washed with phosphate-buffered saline (PBS), and lysed in 1 ml/well MilliQ water that contained protease inhibitors (Roche, Basal, Switzerland). The lysate was centrifuged (5,000 rpm, 5 min, 4°C), and the remaining pellet was resuspended in 100 μL RIPA buffer. The lysate was centrifuged (10,000 rpm, 5 min, 4°C) and the supernatant was transferred to a new collection tube. Protein concentrations were determined by a Pierce BCA protein assay (Pierce, Rockford, IL, USA). Ten micrograms of total protein was separated by SDS–polyacrylamide gel electrophoresis using NuPage novex 4–12% bis–tris gradient gels (Invitrogen, Breda, The Netherlands). Proteins were electroblotted onto Immobilon P membranes (Millipore, Bedford, MA, USA). After blocking (1 h at RT) in 3% BSA in tris-buffered saline containing 0.1% Tween-20, the membranes were incubated overnight at 4°C with a murine monoclonal anti-Pgp primary antibody (C219, 1:500; Abcam, Cambridge, UK) followed by incubation with a goat anti-mouse IgG coupled to horse radish peroxidase (HRP) secondary antibody (1:1,000; ImmunoPure Peroxidase Conjugated Goat anti-Mouse IgG (H + L); Pierce, Rockford, IL, USA). β-actin (1:10,000; AC-15; Abcam, Cambridge, UK) was used as a loading control. The proteins were visualized by a chemiluminescence-based detection reagent (SuperSignal West Femto; Pierce, Rockford, IL, USA), and the intensities of the Pgp bands were determined on a Gel Doc XRS Imaging system with Quantity One analysis software (Bio-Rad, Hercules, CA, USA). The ratio of the Pgp signal and the β-actin signal were indicative for Pgp protein expression.

The efflux activity of Pgp was determined by measuring the accumulation of the fluorescent Pgp probe rhodamine 123 as described by Collett et al. [11]. In brief, LS180 cells were pretreated with the anticancer drugs as described earlier. After 48 h, the cells were washed with Hank’s Balanced Salt Solution (HBSS) and incubated at 37°C for 1 h with rhodamine 123 (10 μM) in the presence or absence of the Pgp-specific inhibitor zosuquidar (LY335979; 5 μM; [12]) in phenol red-free RPMI medium. After washing thrice with ice-cold HBSS, the cells were lysed with Triton X-100 (0.1%) in MilliQ water (Millipore, Bedford, MA, USA). The ratio of intracellular rhodamine 123 concentrations in the absence and presence of 5 μM zosuquidar is indicative for the efflux activity of Pgp.

LS180 cells (0.5 × 10⁶ c/well) were plated in a 24-well plate. After a 48 h incubation with 0.1% DMSO or 10 μM rifampicin, cells were treated with 5 μM doxorubicin for 90 min. Cells were washed thrice with ice-cold PBS, lysed in 250 μl MilliQ water, and homogenized by ultrasonification Labsonic P (Braun; cycle 0.2 min en Amplitude 60%). Hundred microliters of the homogenate was processed for HPLC analysis. Daunorubicin was used as an internal standard. The samples and standard solutions were prepared as described by van Asperen et al. [13].

Doxorubicin was quantified using a method as previously described by DeGregorio et al. [14]. In brief, samples were eluted isocratically with a mobile phase that consisted of 28% acetonitrile in 60 mM phosphoric acid. A Waters Symmetry C18 3.5 μM, 4.6 mm × 100 mm reverse-phase column was used. Fluorescence was detected using a JASCO FP-920 fluorometer (excitation: 455 nm; emission: 550 nm), Shimadzu LC10AT-VP pumps and a Shimadzu SCL10A-VP controller. The flow rate was set at 1.0 ml/min. Doxorubicin and daunorubicin retention times were 2.38 and 5.01 min, respectively. A standard curve of doxorubicin (0.08, 0.4, 2, 10 and 50 μM) was prepared in milliQ water.

LS180 cells were plated (2 × 10⁴ cells/well; 96 well plates) and treated with 10 μM rifampicin or 0.1% DMSO. Following 72 h incubation, cells were exposed to doxorubicin (serially diluted 0–20 μM; dilution factor 2). Cell viability after 48 h doxorubicin exposure was assessed using a neutral red assay [15].