Drug-induced trafficking of p-glycoprotein in human brain capillary endothelial cells as demonstrated by exposure to mitomycin C.

P-glycoprotein (Pgp; ABCB1/MDR1) is a major efflux transporter at the blood-brain barrier (BBB), restricting the penetration of various compounds. In other tissues, trafficking of Pgp from subcellular stores to the cell surface has been demonstrated and may constitute a rapid way of the cell to respond to toxic compounds by functional membrane insertion of the transporter. It is not known whether drug-induced Pgp trafficking also occurs in brain capillary endothelial cells that form the BBB. In this study, trafficking of Pgp was investigated in human brain capillary endothelial cells (hCMEC/D3) that were stably transfected with a doxycycline-inducible MDR1-EGFP fusion plasmid. In the presence of doxycycline, these cells exhibited a 15-fold increase in Pgp-EGFP fusion protein expression, which was associated with an increased efflux of the Pgp substrate rhodamine 123 (Rho123). The chemotherapeutic agent mitomycin C (MMC) was used to study drug-induced trafficking of Pgp. Confocal fluorescence microscopy of single hCMEC/D3-MDR1-EGFP cells revealed that Pgp redistribution from intracellular pools to the cell surface occurred within 2 h of MMC exposure. Pgp-EGFP exhibited a punctuate pattern at the cell surface compatible with concentrated regions of the fusion protein in membrane microdomains, i.e., lipid rafts, which was confirmed by Western blot analysis of biotinylated cell surface proteins in Lubrol-resistant membranes. MMC exposure also increased the functionality of Pgp as assessed in three functional assays with Pgp substrates (Rho123, eFluxx-ID Gold, calcein-AM). However, this increase occurred with some delay after the increased Pgp expression and coincided with the release of Pgp from the Lubrol-resistant membrane complexes. Disrupting rafts by depleting the membrane of cholesterol increased the functionality of Pgp. Our data present the first direct evidence of drug-induced Pgp trafficking at the human BBB and indicate that Pgp has to be released from lipid rafts to gain its full functionality

Effects of the Pgp inhibitor tariquidar (TQ; 0.5 µM) in three functional assays, in which alterations in Pgp efflux are indirectly measured by determining intracellular concentrations of Pgp substrates (Rho123, eFluxx-ID Gold) or their metabolite (calcein AM).

<p>Data are shown as mean ± SEM of three experiments; all experiments with one uptake assay were performed together to avoid inter-experiment variation. In experiments with mitomycin C (MMC) (B,C,D), experiments were performed 20 h after a 4-h exposure to 1 µM MMC. Significant differences between treatments are indicated by asterisk (P<0.01) except for D, in which significance level of the effect of MMC was P = 0.0188 (indicated by rhomb). (A) shows data from the Rho123 uptake assay in nontransfected (wild type) hCMEC/D3 cells in the absence or presence of doxycycline (Dox). Doxycycline (1 µg/ml) did not alter the functionality of Pgp. Tariquidar significantly increased the uptake of Rho123 in wild type cells both in the absence and presence of doxycycline to the same extent. Much higher concentrations (100 µg/ml) of doxycycline have been shown to increase Pgp expression and functionality in MCF-7 breast carcinoma cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088154#pone.0088154-Mealey1" target="_blank">[37]</a>. (B) shows data from the Rho123 uptake assay in transfected hCMEC/D3-MDR1-EGFP cells in the absence and presence of doxycycline. In the absence of doxycycline (open columns), tariquidar significantly increased Rho123 accumulation (i.e., decreased Rho123 efflux) by only about 15%. In the presence of doxycycline, accumulation of Rho123 was only about 50% of that under doxycycline-off conditions, which was completely counteracted by tariquidar. When experiments were performed 20 h after a 4-h exposure to 1 µM MMC, Rho123 accumulation was significantly reduced by 15% under doxycycline-off and 22% under doxycycline-on conditions. Tariquidar counteracted the enhanced functionality of Pgp in response to MMC exposure. However, the tariquidar-induced increase in Rho123 accumulation in MMC-exposed cells remained significantly below the increase seen in the absence of MMC, which was seen both under doxycycline-on and -off conditions. (C) shows data from intracellular accumulation of eFluxx-ID Gold in transfected hCMEC/D3-MDR1-EGFP cells in the presence of doxycycline. In this assay, by using scatter parameters and FL1 (FITC) channel, only viable and EGFP-positive cells were analyzed for intracellular accumulation of the fluorescent Pgp probe. Following MMC exposure, eFluxx-ID Gold accumulation was significantly reduced by 46% under doxycycline-on conditions. Tariquidar completely counteracted the enhanced functionality of Pgp in response to MMC exposure. (D) shows data from intracellular accumulation of calcein in transfected hCMEC/D3-MDR1-EGFP cells in the presence of doxycycline. Following MMC exposure, calcein accumulation was significantly reduced by 10% under doxycycline-on conditions. Tariquidar completely counteracted the enhanced functionality of Pgp in response to MMC exposure.</p

Treatment with mitomycin C (MMC) increases Pgp-EGFP fusion protein at the cell surface.

<p>(A) hCMEC/D3-MDR1-EGFP (doxycycline-on) cells were treated with MMC (1 µM) for 2 or 4 h and analyzed at the end of the 2 h-exposure period or 20 h after the 4 h-exposure period. Western blot analyses of cell surface proteins isolated via EZ-Link Sulfo-NHS-SS-Biotin-Neutravidin assay revealed an obvious increase of Pgp-EGFP abundance at the cell surface after MMC exposure, whereas no effect on intracellular Pgp was seen. One representative result of six independent experiments is shown. In (B), Pgp-EGFP bands of the cell surface were analyzed densitometrically and Pgp signals were normalized relative to the coomassie-stained portion of the gel. Data variability is shown as ± SEM of six experiments; significant differences of treated vs. untreated samples are indicated by asterisk (P<0.05). (C, D) No significant induction of Pgp-EGFP fusion protein was measured in whole cell lysates at the same exposure conditions used in (A). Respective P values in D were 0.5502 (2 h MMC) and 0.2534 (24 h MMC). Pgp-EGFP bands were analyzed densitometrically and Pgp signals were normalized on actin. Data variability is shown as ± SEM of three experiments.</p

Effect of mitomycin C (MMC) on Pgp-EGFP functionality studied with the eFluxx-ID Gold assay in the absence and presence of the Pgp inhibitor tariquidar (TQ; 0.5 µM).

<p>The figure shows data in which eFluxx-ID Gold uptake was measured directly after 2 h of MMC (1 µM) exposure or at 8 and 20 hrs following 4 h of MMC (1 µM) exposure in hCMEC/D3-Pgp-EGFP (doxycyline-on) cells, indicating a lack of MMC exposure on Pgp function in the 2 and 12 h, but not the 24 h experiment. Experiments were performed in triplicates and values are shown as mean ± SEM. Significant differences between control or MMC exposure with tariquidar vs. exposure without tariquidar are indicated by asterisk (P<0.001) while significant difference between MMC exposure (without tariquidar) and control is indicated by rhomb (P = 0.0353).</p

Effect of mitomycin C (MMC) on Pgp-EGFP functionality studied with the Rho123 uptake assay.

<p>MMC significantly increased the functionality of Pgp-EGFP fusion protein. “A”: hCMEC/D3-Pgp-EGFP (doxycycline-off/-on) cells were treated with MMC in Opti-MEM medium (0.1 µM, 1 µM, 10 µM, 50 µM and 100 µM) for 4 hours after which Opti-MEM medium was replaced by complete medium again. Control cells were treated with Opti-MEM medium. After 23 h the cells were incubated with 10 µM Rho123 for 1 h and intracellular accumulation was measured, resulting in a dose-dependent increase in Pgp-EGFP functionality. Experiments were performed in triplicates and values are shown as mean ± SEM. Significant effects of MMC during doxycycline-off conditions are indicated by circles, while significant effects of MMC during doxycycline-on conditions are indicated by rhombs (P<0.05). Significant differences between doxycycline-on and –off conditions are indicated by asterisk (P<0.05). “B” shows data (shown in percent control) in which rhodamine uptake was measured directly after 2 h of MMC (1 µM) exposure or at 4, 8, and 20 hrs following 4 h of MMC (1 µM) exposure in hCMEC/D3-Pgp-EGFP (doxycyline-on) cells, indicating a lack of MMC exposure on Pgp function in the 2, 8 and 12, but not the 24 h experiment. The 24 h experiment was also performed with 10 µM MMC, resulting in similar results (not illustrated). However, when lipid rafts were disrupted by MβCD, MMC (1 µM) exposure for 2 h significantly increased the functionality of Pgp-EGFP fusion protein, which is illustrated by the right columns in “B”. As shown in the inset, this was correlated with a decrease of Pgp in the DRM fraction and an increase of Pgp in the detergent soluble fraction. Significant differences between controls and MMC-treated cells are indicated by asterisk (P<0.05).</p

Schematic illustration of the three experimental strategies used for determining whether exposure to mitomycin C (MMC) induces trafficking of Pgp to the plasma membrane in hCMEC/D3-Pgp-EGFP cells.

<p>Schematic illustration of the three experimental strategies used for determining whether exposure to mitomycin C (MMC) induces trafficking of Pgp to the plasma membrane in hCMEC/D3-Pgp-EGFP cells.</p

Increased Pgp function (as observed in the uptake assays; see Figures 2, 6, and 7) is associated with the transition of Pgp from detergent (Lubrol) resistant membrane (DRM) domains to detergent soluble membrane domains.

<p>Doxycycline-induced hCMEC/D3-MDR1-EGFP cells were treated with MMC (1 µM) for 2 or 4 h and analyzed at the end of the 2 h-exposure period or 20 h after the 4 h-exposure period. Cell surface proteins were biotinylated with EZ-Link Sulfo-NHS-SS-Biotin. After solubilisation of these cells with 1% (w/v) of Lubrol WX, the lysates were centrifuged at 100,000× g for 45 min at 4°C. The DRMs (pellets) resuspended in 0.5 (w/v) DOC and 0.5% (w/v) Triton X-100 and the soluble fractions (supernatant) were subjected to Neutravidin beads to isolate cell surface proteins. These were then analyzed by Western blotting using antibodies against Pgp. The protein bands were analyzed by scanning densitometry. In “A” and “B”, Pgp bands in DRMs and supernatant are presented as percentage of the control in one representative experiment. Data in “C” and “D” are shown as means ± SEM of three experiments. Asterisks denote values that significantly differed (P<0.05).</p

Mitomycin C induced Pgp-EGFP trafficking in hCMEC/D3-MDR1-EGFP (doxycycline-on) cells.

<p>A representative experiment is shown. hCMEC/D3-MDR1-EGFP (doxycycline-on) cells were pretreated with 1 µM MMC for 1 h and cell nuclei of living cells were stained with bisbenzimide H (blue) (scale bars = 30 µm). After 1 h of MMC exposure, fluorescence images were taken every 3.6 min for another 1 h in the presence of MMC. Trafficking events of Pgp-EGFP fusion protein are labeled (arrows). Within 15 min after onset of confocal microscopic analysis (i.e., about 75 min after onset of MMC exposure), we observed that Pgp-EGFP started to traffick from intracellular compartments to the membrane. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088154#pone.0088154.s004" target="_blank">Movie S1</a> illustrating the whole period between 60 min and 139.2 min in fast motion.</p

Conditional doxycycline-dependent Pgp-EGFP-expressing hCMEC/D3-MDR1-EGFP cells.

<p>(A) Representative images showing intracellular localization of Pgp-EGFP fusion protein in hCMEC/D3-MDR1-EGFP (doxycycline-on) cells. Cell nuclei of living cells were stained with bisbenzimide H (blue) (scale bar = 30 µm). (B) Flow cytometric analysis of relative cell size and granularity of hCMEC/D3 wild type, hCMEC/D3-MDR1-EGFP (doxycycline-on) and hCMEC/D3-MDR1-EGFP (doxycycline-off) cells, indicating the lack of any obvious differences between the three conditions; furthermore, as indicated by comparable extent of cell debris, the vitality of the cells does not differ. Along the X-axis is the FSC(Forward SCatter) parameter, while the Y-axis shows the SSC(Side SCatter) parameter. (C,D) Significant Pgp-EGFP fusion protein induction in hCMEC/D3-MDR1-EGFP cells by doxycycline (1 µg/ml) was analyzed by Western blot. Control whole cell lysates of hCMEC/D3 wild type (Wt) and hCMEC/D3-MDR1-EGFP (doxycycline-off/on) cells were used. In C, one representative Western blot of three is shown. Bands were analysed densitometrically and Pgp signals were normalized on actin (D). (E) Induction of Pgp-EGFP by doxycycline (1 µg/ml) significantly decreases Rho123 accumulation in hCMEC/D3-MDR1-EGFP cells confirming that Pgp-EGFP fusion protein is functional. Data in (D) and (E) are shown as mean ± SEM of three experiments. Asterisks denote values that were significantly different from corresponding controls (P<0.05). In all experiments with wild type cells, doxycycline was added for control in order to exclude that doxycycline induces Pgp expression and function also in the absence of an expression vector (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088154#pone-0088154-g002" target="_blank">Figure 2A</a>).</p