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.
References
[1]
Schinkel AH, Jonker JW (2003) Mammalian drug efflux transporters of the ATP binding cassette (ABC) family: an overview. Adv Drug Deliv Rev 55: 3–29. doi: 10.1016/s0169-409x(02)00169-2
[2]
Lee CA, Cook JA, Reyner EL, Smith DA (2010) P-glycoprotein related drug interactions: clinical importance and a consideration of disease states. Expert Opin Drug Metab Toxicol 6: 603–619. doi: 10.1517/17425251003610640
[3]
Hennessy M, Spiers JP (2007) A primer on the mechanics of P-glycoprotein the multidrug transporter. Pharmacol Res 55: 1–15. doi: 10.1016/j.phrs.2006.10.007
[4]
Urquhart BL, Tirona RG, Kim RB (2007) Nuclear receptors and the regulation of drug-metabolizing enzymes and drug transporters: implications for interindividual variability in response to drugs. J Clin Pharmacol 47: 566–578. doi: 10.1177/0091270007299930
[5]
Miller DS (2010) Regulation of P-glycoprotein and other ABC drug transporters at the blood-brain barrier. Trends Pharmacol Sci 31: 246–254. doi: 10.1016/j.tips.2010.03.003
[6]
Fu D, Arias IM (2012) Intracellular trafficking of P-glycoprotein. Int J Biochem Cell Biol 44: 461–464. doi: 10.1016/j.biocel.2013.10.011
[7]
Maitra R, Halpin PA, Karlson KH, Page RL, Paik DY, et al. (2001) Differential effects of mitomycin C and doxorubicin on P-glycoprotein expression. Biochem J 355: 617–624.
[8]
Kipp H, Pichetshote N, Arias IM (2001) Transporters on demand: intrahepatic pools of canalicular ATP binding cassette transporters in rat liver. J Biol Chem 276: 7218–7224. doi: 10.1074/jbc.m007794200
[9]
Fu D, Bebawy M, Kable EP, Roufogalis BD (2004) Dynamic and intracellular trafficking of P-glycoprotein-EGFP fusion protein: Implications in multidrug resistance in cancer. Int J Cancer 109: 174–181. doi: 10.1002/ijc.11659
[10]
Porcelli L, Lemos C, Peters GJ, Paradiso A, Azzariti A (2009) Intracellular trafficking of MDR transporters and relevance of SNPs. Curr Top Med Chem 9: 197–208. doi: 10.2174/156802609787521562
[11]
McCaffrey G, Staatz WD, Sanchez-Covarrubias L, Finch JD, Demarco K, et al. (2012) P-glycoprotein trafficking at the blood-brain barrier altered by peripheral inflammatory hyperalgesia. J Neurochem 122: 962–975. doi: 10.1111/j.1471-4159.2012.07831.x
[12]
L？scher W, Potschka H (2005) Drug resistance in brain diseases and the role of drug efflux transporters. Nature Rev Neurosci 6: 591–602. doi: 10.1038/nrn1728
[13]
Weksler BB, Subileau EA, Perriere N, Charneau P, Holloway K, et al. (2005) Blood-brain barrier-specific properties of a human adult brain endothelial cell line. FASEB J 19: 1872–1874. doi: 10.1096/fj.04-3458fje
[14]
Vutskits GV, Salmon P, Mayor L, Vutskits L, Cudre-Mauroux C, et al. (2006) A role for atm in E-cadherin-mediated contact inhibition in epithelial cells. Breast Cancer Res Treat 99: 143–153. doi: 10.1007/s10549-006-9195-y
[15]
Efferth T, Lohrke H, Volm M (1989) Reciprocal correlation between expression of P-glycoprotein and accumulation of rhodamine 123 in human tumors. Anticancer Res 9: 1633–1637.
[16]
Tai LM, Reddy PS, Lopez-Ramirez MA, Davies HA, Male DK, et al. (2009) Polarized P-glycoprotein expression by the immortalised human brain endothelial cell line, hCMEC/D3, restricts apical-to-basolateral permeability to rhodamine 123. Brain Res 1292: 14–24. doi: 10.1016/j.brainres.2009.10.016
[17]
Ambroziak K, Kuteykin-Teplyakov K, Luna-Tortós C, Al Falah M, Fedrowitz M, L？scher W (2010) Exposure to antiepileptic drugs does not alter the functionality of P-glycoprotein in brain capillary endothelial and kidney cell lines. Eur J Pharmacol 628: 57–66. doi: 10.1016/j.ejphar.2009.11.051
[18]
Dauchy S, Miller F, Couraud PO, Weaver RJ, Weksler B, et al. (2009) Expression and transcriptional regulation of ABC transporters and cytochromes P450 in hCMEC/D3 human cerebral microvascular endothelial cells. Biochem Pharmacol 77: 897–909. doi: 10.1016/j.bcp.2008.11.001
[19]
Lebedeva IV, Pande P, Patton WF (2011) Sensitive and specific fluorescent probes for functional analysis of the three major types of mammalian ABC transporters. PLoS One 6: e22429. doi: 10.1371/journal.pone.0022429
[20]
Huber O, Brunner A, Maier P, Kaufmann R, Couraud PO, et al. (2012) Localization microscopy (SPDM) reveals clustered formations of P-glycoprotein in a human blood-brain barrier model. PLoS One 7: e44776. doi: 10.1371/journal.pone.0044776
[21]
Szakács G, Jakab K, Antal F, Sarkadi B (1998) Diagnostics of multidrug resistance in cancer. Pathol Oncol Res 4: 251–257. doi: 10.1007/bf02905214
[22]
Bauer B, Miller DS, Fricker G (2003) Compound profiling for P-glycoprotein at the blood-brain barrier using a microplate screening system. Pharm Res 20: 1170–1176.
[23]
Neufeld EB, Cooney AM, Pitha J, Dawidowicz EA, Dwyer NK, et al. (1996) Intracellular trafficking of cholesterol monitored with a cyclodextrin. J Biol Chem 271: 21604–21613. doi: 10.1074/jbc.271.35.21604
[24]
Kim RB, Leake BF, Choo EF, Dresser GK, Kubba SV, et al. (2001) Identification of functionally variant MDR1 alleles among European Americans and African Americans. Clin Pharmacol Ther 70: 189–199.
[25]
Sai Y, Nies AT, Arias IM (1999) Bile acid secretion and direct targeting of mdr1-green fluorescent protein from Golgi to the canalicular membrane in polarized WIF-B cells. J Cell Sci 112 (Pt 24): 4535–4545.
[26]
Kipp H, Arias IM (2000) Intracellular trafficking and regulation of canalicular ATP-binding cassette transporters. Semin Liver Dis 20: 339–351. doi: 10.1055/s-2000-9388
[27]
Petriz J, Gottesman MM, Aran JM (2004) An MDR-EGFP gene fusion allows for direct cellular localization, function and stability assessment of P-glycoprotein. Curr Drug Deliv 1: 43–56. doi: 10.2174/1567201043480072
[28]
Vogel M, Hartmann T, Koberle M, Treiber M, Autenrieth IB, et al. (2008) Rifampicin induces MDR1 expression in Candida albicans. J Antimicrob Chemother 61: 541–547. doi: 10.1093/jac/dkm513
[29]
Zhao Z, Liu W, Su Y, Zhu J, Zheng G, et al. (2010) Evaluation of biodistribution and safety of adenovirus vector containing MDR1 in mice. J Exp Clin Cancer Res 29: 1. doi: 10.1186/1756-9966-29-1
[30]
Poller B, Gutmann H, Krahenbuhl S, Weksler B, Romero I, et al. (2008) The human brain endothelial cell line hCMEC/D3 as a human blood-brain barrier model for drug transport studies. J Neurochem 107: 1358–1368. doi: 10.1111/j.1471-4159.2008.05730.x
[31]
Ihnat MA, Nervi AM, Anthony SP, Kaltreider RC, Warren AJ, et al. (1999) Effects of mitomycin C and carboplatin pretreatment on multidrug resistance-associated P-glycoprotein expression and on subsequent suppression of tumor growth by doxorubicin and paclitaxel in human metastatic breast cancer xenografted nude mice. Oncol Res 11: 303–310.
[32]
Ott M, Huls M, Cornelius MG, Fricker G (2010) St. John's Wort constituents modulate P-glycoprotein transport activity at the blood-brain barrier. Pharm Res 27: 811–822. doi: 10.1007/s11095-010-0074-1
[33]
Hartz AM, Bauer B, Fricker G, Miller DS (2004) Rapid regulation of P-glycoprotein at the blood-brain barrier by endothelin-1. Mol Pharmacol 66: 387–394. doi: 10.1124/mol.104.001503
[34]
Kipp H, Arias IM (2002) Trafficking of canalicular ABC transporters in hepatocytes. Annu Rev Physiol 64: 595–608. doi: 10.1146/annurev.physiol.64.081501.155793
[35]
Orlowski S, Martin S, Escargueil A (2006) P-glycoprotein and ‘lipid rafts’: some ambiguous mutual relationships (floating on them, building them or meeting them by chance?). Cell Mol Life Sci 63: 1038–1059. doi: 10.1007/s00018-005-5554-9
[36]
L？scher W, Luna-Tortos C, R？mermann K, Fedrowitz M (2011) Do ATP-binding cassette transporters cause pharmacoresistance in epilepsy? Problems and approaches in determining which antiepileptic drugs are affected. Curr Pharm Des 17: 2808–2828. doi: 10.2174/138161211797440212
[37]
Mealey KL, Barhoumi R, Burghardt RC, Safe S, Kochevar DT (2002) Doxycycline induces expression of P glycoprotein in MCF-7 breast carcinoma cells. Antimicrob Agents Chemother 46: 755–761. doi: 10.1128/aac.46.3.755-761.2002
