Effluxing ABC transporters in human corneal epithelium

Abstract

ATP-binding cassette (ABC) transporters are able to efflux their substrate drugs from the cells. We compared expression of efflux proteins in normal human corneal epithelial tissue, primary human corneal epithelial cells (HCEpiC), and corneal epithelial cell culture model (HCE model) based on human immortal cell line. Expression of multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 1-6 (MRP1-6) and breast cancer resistance protein (BCRP) was studied using quantitative RT-PCR, Western blot, and immunohistochemistry. Only MRP1, MRP5, and BCRP were expressed in the freshly excised human corneal epithelial tissue. Expression of MRP1 and MRP5 was localized predominantly in the basal cells of the central cornea and limbus. Functional efflux activity was shown in the cell models, but they showed overexpression of most efflux transporters compared to that of normal corneal epithelium. In conclusion, MRP1, MRP5, and BCRP are expressed in the corneal epithelium, but MDR1, MRP2, MRP3, MRP4, and MRP6 are not significantly expressed. HCE cell model and commercially available primary cells deviate from this expression profile.

Expression of Efflux Transporters in Human Ocular Tissues

“…Functional activity of some efflux transporters has also been evaluated, but the results of efflux transporter expressions and functions were very different. Dey et al (2003) and Becker et al (2007) investigated the mRNA expression of MDR1 in human corneas, as well as the MDR1 expression in a human corneal cell line by Western blotting, but no detectable or very low MDR1 mRNA and/or protein expressions were observed in human corneas according to other reports (Dey et al, 2003; dmd.aspetjournals.org Becker et al, 2007), and these discrepancies may be due to variation in sensitivity of different experimental methods and different tissues used (Vellonen et al, 2010). Regarding the function of efflux transporters, Dey et al (2003) evaluated the functional activity of MDR1 efflux pump with cultured rabbit primary corneal epithelial cells and a corneal cell line (Statens Seruminstitut rabbit cornea [SIRC] cells) as the model, with the results that MDR1 affected corneal uptake.…”

“…In 2003, Dey et al (2003) found, for the first time, that the human cornea can express MDR1. Since then, individual efflux transporters in human and animal corneas and corneal epithelial cells have been widely investigated (Karla et al, 2007a(Karla et al, ,b, 2009Pelis et al, 2009;Vellonen et al, 2010;Barot et al, 2011;Li et al, 2012). Functional activity of some efflux transporters has also been evaluated, but the results of efflux transporter expressions and functions were very different.…”

“…Moreover, some efflux transporters have been discovered in ocular tissues, such as the cornea and retina (Kajikawa et al, 1999;Kennedy and Mangini, 2002;Becker et al, 2007;Vellonen et al, 2010). Similar to the liver, kidney, and intestine tissues, the expression and function of efflux transporters at various layers of ocular epithelial and endothelial cells may significantly influence ocular drug efficacy by means of absorption, distribution, and elimination (Dey et al, 2004;Hariharan et al, 2009).…”

“…As MRP3 in ICB and RC, and MRP4 in CE had no expression, the statistical analysis was not performed here. Vellonen et al, 2010). However, there have been few reports on the expression pattern of efflux transporters in the other human ocular drug-absorption barrier tissues, such as conjunctiva, iris-ciliary body (ICB), and retina-choroid (RC).…”

The exposure of cancer cells to hyperthermia, iron oxide nanoparticles, and mitomycin C influences membrane multidrug resistance protein expression levels

“…To elucidate the role of MRP 1 and MRP 3 on the protein level, a modified protocol from Vellonen et al 16 was used. Briefly, 7 × 10 6 BT474 cells were suspended in a protein lysis buffer (1% [v/v] Triton X-100, 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA) for 30 minutes on ice and centrifuged for 30 minutes at 10,000 g (4°C) to decant cell detritus and nuclei.…”