Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

Heather M Brechbuhl,Neal Gould,Remy Kachadourian,Wayne R Riekhof,Dennis R Voelker,Brian J Day

doi:10.1074/jbc.m109.090506

Heather M Brechbuhl, Neal Gould + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m109.090506

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Abstract

Glutathione (GSH) transport is vital for maintenance of intracellular and extracellular redox balance. Only a few human proteins have been identified as transporters of GSH, glutathione disulfide (GSSG) and/or GSH conjugates (GS-X). Human epithelial MDA1586, A549, H1975, H460, HN4, and H157 cell lines were exposed to 2',5'-dihydroxychalcone, which induces a GSH efflux response. A real-time gene superarray for 84 proteins found in families that have a known role in GSH, GSSG, and/or GS-X transport was employed to help identify potential GSH transporters. ABCG2 was identified as the only gene in the array that closely corresponded with the magnitude of 2',5'-dihydroxychalcone (2',5'-DHC)-induced GSH efflux. The role of human ABCG2 as a novel GSH transporter was verified in a Saccharomyces cerevisiae galactose-inducible gene expression system. Yeast expressing human ABCG2 had 2.5-fold more extracellular GSH compared with those not expressing ABCG2. GSH efflux in ABCG2-expressing yeast was abolished by the ABCG2 substrate methotrexate (10 microM), indicating competitive inhibition. In contrast, 2',5'-DHC treatment of ABCG2-expressing yeast increased extracellular GSH levels in a dose-dependent manner with a maximum 3.5-fold increase in GSH after 24 h. In addition, suppression of ABCG2 with short hairpin RNA or ABCG2 overexpression in human epithelial cells decreased or increased extracellular GSH levels, respectively. Our data indicate that ABCG2 is a novel GSH transporter.

Highlights

Mented [3,4,5,6,7,8]
Model Results in Increased GSH Efflux—Human ABCG2 was expressed in the yeast strain S. cerevisiae mutant for the yeast
Our results demonstrate a novel GSH transport function for ABCG2

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Treatment—All cell lines were cultured at 37 °C with 5% CO2. Human epithelial lung cancer cell lines A549, H157, H460, and H1975 were grown in RPMI 1640 medium supplemented with L-glutamine (Mediatech, Manassas, VA) and 5% fetal bovine serum (Gemini Bio-Products, West Sacramento, CA). Cell lysate protein values were used to normalize GSH values and were determined spectrophotometrically with the Coomassie Protein Assay kit according to the manufacturer’s protocol (Pierce). ABCG2 silencing in MDA1586 cells was achieved by inserting the ABCG2 shRNA target sequence (NM_004827.2) into pSilencer 4.1-CMV hygro vector (Applied Biosystems/Ambion, Austin, TX) Both plasmids were transfected into the appropriate cell model with Effectene Transfection Reagent according to the manufacturer’s protocol (Qiagen, Valencia, CA). Real-time Reverse Transcriptase (SYBR Green) SuperArray Assay—Isolation of mRNA from cells grown in a T-75 was completed using the Qiagen RNeasy Mini Spin ColumnQIAshredder kit (Qiagen), according to the manufacturer’s protocol. Two-stage real-time reverse transcriptase PCR of ABC family members and several GSH and SLC family members was performed on a custom designed 96-well format RT2 SuperArray (SABiosciences). One-way analysis of variance (ANOVA) and Tukey post comparison test or Student’s t test were performed using Prizm version 5 (GraphPad, San Diego, CA)

RESULTS

DISCUSSION

GSH efflux may provide a pharmaceutical approach for increasing