Influence of tumor necrosis factor-α on the expression and function of P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT

doi:10.1016/S0006-2952(03)00340-X

Biochemical Pharmacology

Volume 66, Issue 4, 15 August 2003, Pages 579-587

https://doi.org/10.1016/S0006-2952(03)00340-X Get rights and content

Abstract

Drug cerebral pharmacokinetics may be altered in the case of inflammatory diseases. This may be due to a modification of drug transport through the blood–brain barrier, in particular through drug interaction with the membrane efflux transporter, P-glycoprotein. The objective of this study was to investigate the influence of the inflammatory cytokine, tumor necrosis factor (TNF)-α, on the functionality and expression of P-glycoprotein, and on mdr1a and mdr1b mRNA expression in immortalised rat brain endothelial cells, GPNT. Cells were treated with TNF-α for 4 days. Levels of mdr1a and mdr1b mRNAs were quantitated using real-time RT-PCR analysis and expression of P-glycoprotein was analyzed by Western blot. The functionality of P-glycoprotein was studied by following the accumulation of [ $^{3} H$ ]vinblastine in the cells without and with a pre-treatment with a P-glycoprotein inhibitor, GF120918. TNF-α increased the levels of mdr1a and mdr1b mRNAs while no effect was observed on protein expression. TNF-α increased [ $^{3} H$ ]vinblastine accumulation indicating a time and concentration-dependent decrease of P-glycoprotein activity. This effect was eliminated when the cells were pre-treated with GF120918. Our observation of a decrease in P-glycoprotein activity could suggest that in the case of inflammatory diseases, brain delivery of P-glycoprotein-dependent drugs can be enhanced.

Introduction

P-gp is an efflux plasma membrane protein overexpressed in tumor cells and acting as a pump, effluxing anticancer drugs from the cells. It confers to these cells a multidrug resistance. It is also expressed in non-malignant tissues such as lung, intestine, kidney, epithelia, testis and brain capillary endothelium [1], [2], [3]. P-gp is encoded by a gene family comprising two mdr genes (MDR1 and MDR2) in humans and three mdr genes (mdr1a, mdr1b and mdr2) in rodents [4], respectively, hABCB1 and hABCB2 in humans and rAbcb1a, rAbcb1b and rAbcb2 in rodents according to the new “nomenclature.” However, only the expression of human MDR1 and rodent mdr1a and mdr1b appears to selectively confer multidrug resistance. In the endothelium of rat brain capillary, mdr1a is exclusively expressed and the P-gp mdr1a isoform contributes solely to the efflux of drugs from the brain back into the blood circulation. Experiments with knock-out mice deficient in mdr1a have shown the role of mdr1a and P-glycoprotein in the brain uptake of many drugs with potential targets in the central nervous system [5], [6].

Central nervous system (CNS) inflammatory pathologies such as multiple sclerosis, bacterial and fungal meningitis, Alzheimer’s disease and AIDS dementia complex may modify the integrity of the BBB. Inflammatory cytokines (TNF-α; interleukin-1β, IL1β; and interleukin-6, IL6) are predominantly secreted into the CNS after injury or inflammation by macrophages, microglial cells, astrocytes and capillary endothelial cells [7], [8], [9].

Contradictory studies have reported that inflammatory cytokines could modify the expression and the functionality of P-gp. Cellular accumulation of doxorubicin was increased by TNF-α in Caco-2 cells suggesting a decrease in P-gp functionality [10], while rhodamine 123 accumulation was decreased by TNF-α in primary rat hepatocyte culture indicating an increased functionality of P-gp [11].

At the BBB level, Mandi et al. [12] observed that TNF-α (10³ U/mL) did not influence the expression of P-gp and seemed to decrease its functionality on human BB19 brain capillary endothelial cells. However, no statistics were applied to these data and the expression of mdr1a and mdr1b mRNAs was not investigated.

The objective of our study was to investigate the modification of the expression and functionality of P-gp by different concentrations of TNF-α in an immortalised rat brain capillary endothelial cell line, GPNT. Cells were treated with TNF-α at 0.1, 1, and 10 ng/mL for 2–96 hr. We have studied the TNF-α effect on the expression of mdr1a and mdr1b mRNA and P-gp protein, and we have measured the intracellular accumulation of [ $^{3} H$ ]vinblastine with or without the P-gp inhibitor, GF120918.

Section snippets

Materials

Media and foetal calf serum (FCS) of USA origin were obtained from Life Technologies. [ $^{3} H$ ]vinblastine sulfate (8.1 Ci/mmol) was purchased from Amersham Pharmacia Biotech. The P-gp inhibitor GF120918 (GF120918) was provided by Glaxo SmithKline. GF120918 was dissolved in dimethylsulfoxide (DMSO) with a final DMSO concentration in the cell incubation media of 0.01% (v/v). Rat recombinant TNF-α was purchased from R&D Systems. The chemiluminescence detection kit (ECL) was purchased from Pierce and

Cell viability after TNF-α treatment

After treatment with 0.1 and 10 ng/mL TNF-α for 48 and 96 hr, cell viability was not modified (within 10% of control).

RT-PCR analysis

To establish mdr1a and mdr1b gene expression production by GPNT cell line in response to TNF-α treatment, amplification of cDNA was assessed. To quantify the relative abundance of the mdr1a and mdr1b transcripts, we performed real-time PCR using specific primers (Table 1). We obtained the slopes of the standard curves (threshold cycle vs. log[amplicon]) that gave values of 3 with

Discussion

TNF-α induced an increase in mdr1a and mdr1b mRNA expression, whereas no change in the protein level was observed. Moreover, treatments with every tested TNF-α concentration resulted in a P-gp activity decrease, which could not be correlated with any change in P-glycoprotein expression.

Conclusion

All these apparent discrepancies in the regulation of mdr mRNA expression, protein expression, and P-gp activity indicate that different results may be obtained depending on the species, the type of cell model, the procedures of culture used and the treatment protocols. Indeed, the numerous mechanisms involved in these processes (such as transcriptional and translational regulations, mRNA stability, phosphorylation state of the protein and changes of it physico-chemical properties) could

Acknowledgements

This work was supported by a grant from Janssen Cilag laboratories (Issy-les-moulineaux, France) and by the Institut de la Santé et de la Recherche Médicale (INSERM).

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Influence of tumor necrosis factor-α on the expression and function of P-glycoprotein in an immortalised rat brain capillary endothelial cell line, GPNT

Abstract

Introduction

Section snippets

Materials

Cell viability after TNF-α treatment

RT-PCR analysis

Discussion

Conclusion

Acknowledgements

Expression of a multidrug-resistance gene in human tumors and tissues

Proc. Natl. Acad. Sci. U.S.A.

Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and cross reactivity of one antibody with a muscle protein

J. Histochem. Cytochem.

Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues

J. Histochem. Cytochem.

Identification of members of the P-glycoprotein multigene family

Mol. Cell. Biol.

Absence of the mdr1a P-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A

J. Clin. Invest.

P-glycoprotein in the blood–brain barrier of mice influences the brain penetration and pharmacological activity of many drugs

J. Clin. Invest.

The influence of cytokines on the integrity of the blood–brain barrier in vitro

J. Neuroimmunol.

The blood–brain barrier in neuroinflammatory diseases

Pharmacol. Rev.

Effect of tumor necrosis factor on receptor-mediated endocytosis and barrier functions of bovine brain capillary endothelial cell monolayers

J. Neuroimmunol.

Modulation of mdr1 expression by cytokines in human colon carcinoma cells: an approach of multidrug resistance

Br. J. Cancer

Induction of mdr1b mRNA and P-glycoprotein expression by tumor necrosis factor alpha in primary rat hepatocyte cultures

J. Cell. Physiol.

Nitric oxide production and MDR expression by human brain endothelial cells

Anticancer Res.

SV40 large T immortalised cell lines of the rat blood–brain and blood–retinal barriers retain their phenotypic and immunological characteristics

J. Neuroimmunol.

Dexamethasone regulation of P-glycoprotein activity in an immortalised rat brain endothelial cell line, GPNT

J. Neurochem.

Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction

Anal. Biochem.

HIV-1-induced production of endothelin-1 in an in vitro model of the human blood–brain barrier

Neuroreport