Exposure of LS-180 cells to drugs of diverse physicochemical and therapeutic properties up-regulates P-glycoprotein expression and activity

Abstract

Purpose: Drug transporters are increasingly recognized as important determinants of variability in drug disposition and therapeutic response, both in pre-clinical and clinical stages of drug development process. The role P-glycoprotein (P-gp) plays in drug interactions via its inhibition is well established. However, much less knowledge is available about drugs effect on P-gp up-regulation. The objective of this work was to in vitro investigate and rank commonly used drugs according to their potencies to up-regulate P-gp activity utilizing the same experimental conditions.

Methods: The in vitro potencies of several drugs of diverse physicochemical and therapeutic properties including rifampicin, dexamethasone, caffeine, verapamil, pentylenetetrazole, hyperforin, and β-estradiol over broad concentration range to up-regulate P-gp expression and activity were examined. For dose-response studies, LS-180 cells were treated with different concentrations of the selected drugs followed by P-gp protein and gene expressions analyses. P-gp functionality was determined by uptake studies with rhodamine 123 as a P-gp substrate, followed by Emax/EC50 evaluation.

Results: The results demonstrated a dose-dependent increase in P-gp expression and activity following treatments. At 50 uM concentration (hyperforin, 0.1 uM), examined drugs increased P-gp protein and gene expressions by up to 5.5 and 6.2-fold, respectively, while enhanced P-gp activity by 1.8-4-fold. The rank order of these drugs potencies to up-regulate P-gp activity was as following: hyperforin >>> dexamethasone ~ beta-estradiol > caffeine > rifampicin ~ pentylenetetrazole > verapamil.

Conclusions: These drugs have the potential to be involved in drug interactions when administered with other drugs that are P-gp substrates. Further studies are needed to in vivo evaluate these drugs and verify the consequences of such induction on P-gp activity for in vitro-in vivo correlation purposes.

Figure 1

Chemical structures for the investigated drugs.

Figure 2

Representative Western blots for P-gp in LS-180 cells treated with rifampicin, verapamil, dexamethasone, and hyperforin. Cells were treated for 48 h with increasing concentrations of the indicated drugs in the range of 5–100 μM except for hyperforin (25–150 nM). P-gp protein expression was analyzed by immunoblotting with C-219 primary antibody.

Figure 3

Effect of treatment of LS-180 cells with increasing concentrations of (A) rifampicin, (B) verapamil, (C) dexamethasone, (D) pentylenetetrazole, (E) β-estradiol, (F) hyperforin, and (G) caffeine, on P-gp expression and activity. Cells were treated for 48 h followed by Western blot analysis for P-gp expression (shown as column bars) and uptake study for P-gp activity (shown as line). P-gp activity was measured as the ratio of intracellular rhodamine 123 fluorescence in the presence and absence of 100 μM verapamil as P-gp inhibitor. The data is expressed as mean ± STD for P-gp activity (n=3–4). P-gp expression was determined for n=2. *P < 0.05.

Figure 4

Effect of treatment with rifampicin (RIF), dexamethasone (DEX), verapamil (VER), hyperforin (HYP), β-estradiol (EST), pentylenetetrazole (PTZ), and caffeine (CAF) on P-gp expression, MDR1 mRNA expression and activity in LS-180 cells compared to control (CTRL). The drugs indicated were incubated with LS-180 cells at 50 μM for 48 h. P-gp protein expression was analyzed using Western blotting, MDR1 mRNA was quantified using real-time PCR analysis, and P-gp activity was estimated utilizing uptake studies utilizing rhodamine 123 as P-gp substrate (1 μg/ml). The data is expressed as mean ± STD for MDR1 mRNA expression (n=3) and P-gp activity (n=3–4). P-gp expression was determined for n=2. *P < 0.05.