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Reduction in ABCG2 mRNA Expression in Human Immortalised Brain Microvascular Endothelial Cells by Ferric Ammonium Citrate is Mediated by Reactive Oxygen Species and Activation of ERK1/2 Signalling

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Abstract

Purpose

The ATP-binding cassette (ABC) transport protein ABCG2 (also known as breast cancer resistance protein (BCRP)) is expressed at the luminal face of the blood-brain barrier (BBB), where it limits the brain uptake of a number of therapeutic drugs. We recently reported that the ABC efflux transporter P-glycoprotein (P-gp) was downregulated in human immortalised brain endothelial (hCMEC/D3) cells treated with ferric ammonium citrate (FAC). The aim of the present study, therefore, was to assess whether BCRP expression is also affected by FAC and identify any signalling mechanisms involved.

Methods

ABCG2 mRNA was assessed by RT-qPCR. Protein levels of BCRP, phosphorylated extracellular-regulated kinases 1 and 2 (p-ERK1/2) and total ERK 1/2 were assessed by Western blot. Reactive oxygen species (ROS) levels were determined using 2′,7′-dichlorofluorescin diacetate.

Results

Treatment of hCMEC/D3 cells with FAC (250 µM, 72 h) significantly reduced ABCG2 mRNA levels (32.2 ± 3.7%) without a concomitant reduction in BCRP protein expression. ABCG2 mRNA levels were restored to control levels when co-treated with the antioxidant N-acetylcysteine (NAC), suggesting the effect of FAC was mediated by a ROS-sensitive pathway. We also found that FAC-treatment was associated with increased levels of p-ERK1/2, suggesting involvement of the ERK1/2 signalling pathway in the observed ABCG2 mRNA downregulation. The ERK1/2 signalling pathway inhibitor U0126 restored p-ERK1/2 levels and partially attenuated the FAC-induced reduction in ABCG2 mRNA.

Conclusions

This study suggests that FAC-induced downregulation of ABCG2 mRNA is driven by ROS and ERK1/2 signalling, mechanisms which may be exploited to modulate BCRP expression at the BBB.

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Data Availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

Aβ:

β-amyloid

AD:

Alzheimer’s disease

BBB:

Blood-brain barrier

BCRP/ABCG2:

Breast cancer resistance protein

DCFH-DA:

2′,7′-Dichlorofluorescin diacetate

FAC:

Ferric ammonium citrate

hCMEC/D3:

Immortalised human cerebral microvascular endothelial cell line

NAC:

N-acetylcysteine

p-ERK1/2:

Phosphorylated extracellular-regulated kinases 1 and 2

P-gp:

P-glycoprotein

ROS:

Reactive oxygen species

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Funding

The work presented in this manuscript was funded by the Mason Foundation National Medical Program (MAS2017F035). Stephanie Newman was supported by a ‘Research Training Program’ stipend, provided by the Australian Government.

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Authors and Affiliations

  1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia

    Stephanie A. Newman & Joseph A. Nicolazzo

  2. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australia

    Jennifer L. Short

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  1. Stephanie A. Newman
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Correspondence to Joseph A. Nicolazzo.

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Newman, S.A., Short, J.L. & Nicolazzo, J.A. Reduction in ABCG2 mRNA Expression in Human Immortalised Brain Microvascular Endothelial Cells by Ferric Ammonium Citrate is Mediated by Reactive Oxygen Species and Activation of ERK1/2 Signalling. Pharm Res 40, 651–660 (2023). https://doi.org/10.1007/s11095-022-03458-w

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  • DOI: https://doi.org/10.1007/s11095-022-03458-w

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