TY - JOUR
T1 - Development and validation of an in-cell western for quantifying P-glycoprotein expression in human brain microvascular endothelial (hCMEC/D3) cells
AU - McInerney, Mitchell P
AU - Pan, Yijun
AU - Short, Jennifer L.
AU - Nicolazzo, Joseph A.
PY - 2017/9
Y1 - 2017/9
N2 - An in-cell western (ICW) protocol detecting the relative expression of P-glycoprotein (P-gp) in human cerebro-microvascular endothelial cells (hCMEC/D3) was developed and optimized, with the intention of improving throughput relative to western blotting (WB). For validation of the ICW protocol, hCMEC/D3 cells were incubated with known P-gp upregulators (10 μM rifampicin and 5 μM SR12813) and treated with siRNA targeted against MDR1, before measuring changes in P-gp expression, using both ICW and WB in parallel. To confirm a relationship between the detected P-gp expression and function, the uptake of the P-gp substrate rhodamine-123 was assessed following SR12813 treatment. Rifampicin and SR12813 significantly upregulated P-gp expression (1.5-fold and 1.9-fold, respectively) compared to control, as assessed by the ICW protocol. WB analysis of the same treatments revealed 1.4-fold and 1.5-fold upregulations. MDR1 siRNA reduced P-gp abundance by 20% and 35% when assessed by ICW and WB, respectively. SR12813 treatment reduced rhodamine-123 uptake by 18%, indicating that the observed changes in P-gp expression by ICW were associated with comparable functional changes. The correlation of P-gp upregulation by WB, rhodamine-123 uptake, and the ICW protocol provide validation of a new ICW method as an alternative method for quantification of P-gp in hCMEC/D3 cells.
AB - An in-cell western (ICW) protocol detecting the relative expression of P-glycoprotein (P-gp) in human cerebro-microvascular endothelial cells (hCMEC/D3) was developed and optimized, with the intention of improving throughput relative to western blotting (WB). For validation of the ICW protocol, hCMEC/D3 cells were incubated with known P-gp upregulators (10 μM rifampicin and 5 μM SR12813) and treated with siRNA targeted against MDR1, before measuring changes in P-gp expression, using both ICW and WB in parallel. To confirm a relationship between the detected P-gp expression and function, the uptake of the P-gp substrate rhodamine-123 was assessed following SR12813 treatment. Rifampicin and SR12813 significantly upregulated P-gp expression (1.5-fold and 1.9-fold, respectively) compared to control, as assessed by the ICW protocol. WB analysis of the same treatments revealed 1.4-fold and 1.5-fold upregulations. MDR1 siRNA reduced P-gp abundance by 20% and 35% when assessed by ICW and WB, respectively. SR12813 treatment reduced rhodamine-123 uptake by 18%, indicating that the observed changes in P-gp expression by ICW were associated with comparable functional changes. The correlation of P-gp upregulation by WB, rhodamine-123 uptake, and the ICW protocol provide validation of a new ICW method as an alternative method for quantification of P-gp in hCMEC/D3 cells.
KW - ABC transporters
KW - Blood-brain barrier
KW - Cell culture
KW - Drug resistance
KW - Efflux pumps
KW - Endothelial
KW - Membrane transporter
KW - P-glycoprotein
UR - http://www.scopus.com/inward/record.url?scp=85011117095&partnerID=8YFLogxK
U2 - 10.1016/j.xphs.2016.12.017
DO - 10.1016/j.xphs.2016.12.017
M3 - Article
C2 - 28065764
AN - SCOPUS:85011117095
SN - 0022-3549
VL - 106
SP - 2614
EP - 2624
JO - Journal of Pharmaceutical Sciences
JF - Journal of Pharmaceutical Sciences
IS - 9
ER -