Drug-Induced Trafficking of P-Glycoprotein in Human Brain Capillary Endothelial Cells as Demonstrated by Exposure to Mitomycin C

Abstract

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.

P-glycoprotein traffics from the nucleus to the plasma membrane in rat brain endothelium during inflammatory pain

“…The acute, 3 h, timepoint makes it unlikely that de novo PgP synthesis accounts for our observations. 13 PgP has been found in the nuclear envelope in BBB endothelial cells in vivo by electron microscopy 14 consistent with our observations in the current study. In some tumor cells, PgP is glycosylated and active in the nuclear membrane where it functions to keep drugs (particularly doxorubicin) out of the nucleus, thereby contributing to tumor cell survival.…”

Regulation of breast cancer resistance protein and P-glycoprotein by ezrin, radixin and moesin in lung, intestinal and renal cancer cell lines

“…We conducted efflux assays and evaluated efflux rates as previously reported . Briefly, cells were washed twice using cold Dulbecco's phosphate‐buffered saline (PBS) and the uptake process was started in cold Opti‐MEM containing 5 μ m SN‐38 or 10 μ m Rho123 on ice.…”

“…We conducted efflux assays and evaluated efflux rates as previously reported. [23,24,28] Briefly, cells were washed twice using cold Dulbecco's phosphate-buffered saline (PBS) and the uptake process was started in cold Opti-MEM containing 5 lM SN-38 or 10 lM Rho123 on ice. After 30 min, cells were washed twice with cold PBS and incubated with 300 ll Opti-MEM at 37°C for 10 min.…”

A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier

“…However, whether P-gp constantly circulates in cells and is recruited to the cell membrane upon its encounter with its substrate in the cytoplasm or persists on the cell membrane and pumps out its substrates into the extracellular space is not clearly proven. In a recent report, mitomycin C treatment induced the translocation of the cytoplasmic portion of P-gp to the cell surface, suggesting that relocalization of P-gp can be induced by chemical triggers (41). Many drugs developed for treatment of brain disorders are largely classified as P-gp substrates; thus, significant efforts are placed on developing methods to bypass the hindrance posed by P-gp (27,42,43).…”