A Combination of Clioquinol, Zinc and Copper Increases the Abundance and Function of Breast Cancer Resistance Protein in Human Brain Microvascular Endothelial Cells

Abstract

Modulating the abundance of the blood-brain barrier (BBB) efflux transporter breast cancer resistance protein (BCRP) has the potential to impact brain levels of drugs and endogenous substrates. Studies have demonstrated that the metal ionophore clioquinol (CQ) increases BBB abundance of P-glycoprotein (Pgp), an effect associated with increased endothelial cell levels of Cu 2þ . This study therefore assessed whether human brain endothelial (hCMEC/D3) cell abundance and function of BCRP is modulated by CQ. hCMEC/D3 cells were treated with CQ, Zn 2þ and Cu 2þ (CZC) (0.5 mM, 0.5 mM, 0.1 mM, respectively) for 24 h and BCRP mRNA and protein abundance was determined by Western blot and qPCR, respectively. After a series of optimisation studies assessing specificity of bodipy prazosin (BP) and Ko143 as a substrate and inhibitor of BCRP, respectively, the impact of CZC on BP uptake was assessed. While CZC did not increase mRNA expression of BCRP, BCRP abundance was increased 1.8 ± 0.1-fold; this was associated with a 68.1 ± 3.3% reduction in accumulation of BP in hCMEC/D3 cells. This is the first study to demonstrate that augmenting metal ion availability enhances protein abundance and function of BCRP at the BBB, which may be exploited to modulate CNS access of therapeutics and endogenous substrates.

Cu(ATSM) Increases P-Glycoprotein Expression and Function at the Blood-Brain Barrier in C57BL6/J Mice

“…The identification of potential modulators of P-gp expression and function, and understanding the regulatory mechanisms involved in maintaining the function of this gatekeeper, has numerous applications in developing neurotherapeutics. Copper influences pathways involved in P-gp regulation, and our previous findings showed modulation of key efflux transporters in hCMEC/D3 cells treated with copper-modulating compounds [ 36 , 40 , 50 , 51 ]. Therefore, this study investigated the impact of Cu(ATSM) in mBECs and the mouse microvasculature in vivo to determine if the observed changes in hCMEC/D3 cells translated to mice, where Cu(ATSM) has been shown to exhibit disease-reversing effects.…”