Effects of dabrafenib on the activation of human, mouse and zebrafish pregnane X receptor (PXR) and human constitutive androstane receptor (CAR) nuclear receptors as measured by luciferase reporter assay. Activation has been measured in (A) HG5LN control cells or cells expressing GAL4 fusion with (B) human (hPXR), (C) mouse (mPXR) or (D) zebrafish (zfPXR) PXR, (E) human wild-type (hCAR) or (F) mutated (hCAR APYLT) hCAR LBD treated by DMSO (0.1%), SR12813 3 μM, PCN 3 μM, clotrimazol 1 μM, CITCO 1 μM and dabrafenib 3 μM. Results are expressed as fold induction as compared to control. Data are expressed as the mean ± sd of three independent experiments, **** p < 0.0001, *** p < 0.001, ** p < 0.01 (Student’s t-test) compared with DMSO-treated cells.

Dabrafenib is a potent activator of hPXR on different human cancer cell lines. Results of luciferase assays showing dose–response curves for SR12813 and dabrafenib in (A) HG5LN GAL4-hPXR, (B) LS174T-hPXR, (C) HepG2-hPXR and (D) 22RV1-hPXR reporter cell lines. Results are expressed as a percentage of the maximal response obtained in the presence of 3 μM SR12813. Data are the mean ± sd of three to five independent experiments, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.1 (Student’s t-test) compared with DMSO-treated cells.3.2. Time-Resolved Fluorescence Energy Transfer Experiments.

Dabrafenib binds to hPXR. Inhibition of FRET between fluorescein-labeled PXR ligand and recombinant GST-hPXR by SR12813 or dabrafenib. Results are expressed as the signal from the fluorescein emission divided by the terbium signal to provide a TR-FRET emission ratio. Data are the mean (±sd) from three independent experiments, *** p < 0.001, ** p < 0.01, * p < 0.1 (Student’s t-test) compared with DMSO-treated cells.3.3. Induction of hPXR-Mediated Proliferation by Dabrafenib.

Effects of dabrafenib and SR12813 on the proliferation of LS174T-hPXR cells. (A). LS174T control cells and LS174T hPXR cells were treated with 0.1% DMSO, 3 μM SPA70, 0.3 μM SR12813, 0.3 μM SR12813 + 3 μM SPA70, 0.3 μM dabrafenib and 0.3 μM dabrafenib + 3 μM SPA70 for 7 days. (B) LS174T hPXR cells were pretreated 24 h with SPA70 100 nM. This pretreatment was performed in order to reduce the basal proliferation of the cells, thus allowing a more sensitive detection of any PXR agonist activity that would result in growth induction. Then, medium was removed, cells were treated with increasing concentrations of SR12813 or dabrafenib for 7 days continuously, and cell growth was measured using MTT assay. Data are expressed as fold change in cell growth as compared to untreated cells and expressed as mean ± sd of three to six independent experiments, **** p < 0.0001, *** p < 0.001 (Student’s t-test) compared to DMSO-treated cells.

Effects of dabrafenib on hPXR target genes expression in LS174T-hPXR cells and in primary human hepatocytes. (A) RT-qPCR of CYP34A, ABCG2 and FGF19 mRNA expression were performed in LS1747-hPXR cells treated with 3 μM SR12813 or dabrafenib for 24 h. Results were obtained from three independent experiments performed in duplicate. Data are expressed as mean ± sd and compared with control cells treated with DMSO (0.1%). (B) RT-qPCR of CYP34A and CYP2B6 mRNA expression in primary cultures of human hepatocytes (three independent donors: FT438, FH439 and FH440) following 48 h treatment with the indicated concentrations of ligand. The relative gene expression levels were normalized using GAPDH content for LS174T-hPXR cells and for RPLP0 for human hepatocytes. mRNA expressions are expressed as mean ± sd of three independent experiments and compared with DMSO-treated LS174T-hPXR cells or hepatocytes, **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.1 (Student’s t-test).