Mechanism of drug extrusion by brain endothelial cells via lysosomal drug trapping and disposal by neutrophils

Abstract

SignificanceLocated at the apical (blood-facing) site of brain capillary endothelial cells that form the blood–brain barrier (BBB), the efflux transporter P-glycoprotein (Pgp) restricts the brain entry of various lipophilic xenobiotics, which contributes to BBB function. Pgp may become saturated if exposed to too-high drug concentrations. Here, we demonstrate a second-line defense mechanism in human brain capillary endothelial cells—that is, Pgp-mediated intracellular lysosomal drug trapping. Furthermore, we describe a mechanism of drug disposal at the BBB, which is shedding of lysosomal Pgp/substrate complexes at the apical membrane of human and porcine BBB endothelial cells and subsequent phagocytosis by neutrophils. Thus, we have discovered a fascinating mechanism of how Pgp might contribute to brain protection.

TFEB-mediated lysosomal biogenesis and lysosomal drug sequestration confer resistance to MEK inhibition in pancreatic cancer

“…Irrespective of their acid-base properties, cells may sequester certain xenobiotics into the lysosomes as a detox mechanism. In fact, lysosomal drug sequestration plays a protective role in brain cells where lysosomes trap drugs and deposit them in neutrophils by extrusion 35 . Malignant cancer cells may adopt such mechanisms to guard against therapeutic assaults.…”

Novel Intrinsic Mechanisms of Active Drug Extrusion at the Blood-Brain Barrier: Potential Targets for Enhancing Drug Delivery to the Brain?

“…Prompted by Pgp-mediated lysosomal trapping in cancer cells, we recently studied if the lysosomal sequestration of Pgp substrates, including doxorubicin, also occurs in BCECs that form the BBB [ 143 ]. As in our previous studies on intra- and intercellular Pgp trafficking, MDR1 -EGFP transduced hCMEC/D3 cells were used as a model.…”

“…Our experiments demonstrated that the endolysosomal trapping of Pgp substrates occurs in human and porcine BCECs ( Figure 1 G, Figure 3 (5), Figure 5 ) [ 143 ]. Unexpectedly, following endolysosomal drug sequestration, we observed subsequent shedding of these Pgp substrate-sequestering vesicular structures ( Figure 3 (6)).…”

“…The localization of barrier-body aggregates at the plasma membrane border of adjacent endothelial cells and the presence of both the WT marker eFluor670 and Pgp-EGFP in the barrier bodies indicated that each barrier-body aggregate (which was exhibited by ≈10% of the endothelial cells) was formed by vesicles from more than one endothelial cell. This suggests that at least 20% of the cells in the cocultures of hCMEC/D3 MDR1 -EGFP and hCMEC/D3 WT cells contributed to barrier-body formation [ 143 ]. The same processes of lysosomal drug sequestration, barrier-body formation and phagocytosis by neutrophils were also observed in primary cultured porcine BCECs [ 143 ] ( Figure 6 ).…”

“…This suggests that at least 20% of the cells in the cocultures of hCMEC/D3 MDR1 -EGFP and hCMEC/D3 WT cells contributed to barrier-body formation [ 143 ]. The same processes of lysosomal drug sequestration, barrier-body formation and phagocytosis by neutrophils were also observed in primary cultured porcine BCECs [ 143 ] ( Figure 6 ). Thus, barrier bodies are not a non-physiological phenomenon (or artifact) that occurr only in MDR1 -EGFP transduced hCMEC/D3 cells, but they also occurred in WT hCMEC/D3 cells that received Pgp-EGFP from donor cells and in primary cultures of pBCECs.…”