Aryl hydrocarbon receptor is a transcriptional activator of the human breast cancer resistance protein (BCRP/ABCG2).

Breast cancer resistance protein (BCRP/ABCG2) is a membrane-bound efflux transporter important in cellular detoxification and multidrug resistance. Some aryl hydrocarbon receptor (AHR) agonists were reported to induce BCRP expression in human colon carcinoma cells. However, a direct involvement of AHR transcriptional regulation remains unexplored. In this study, we show that BCRP induction by AHR ligands occurs in human intestinal, liver, and mammary carcinoma cells and in primary colonocytes and hepatocytes. Increased BCRP transporter activity consistent with gene induction was also evident in the Caco2 subclone C2bbe1 cells. Using RNA interference and ectopic expression techniques to manipulate cellular AHR status, we confirmed AHR dependence of ABCG2 gene regulation. By gene promoter analysis, chromatin immunoprecipitation, and electrophoretic mobility shift assays, an active, proximal dioxin-response element at -194/-190 base pairs upstream of the transcription start site of the human ABCG2 gene was identified. Despite a common observation in human-derived cells, our in vitro and in vivo studies supported by phylogenetic footprinting analysis did not find that mouse Abcg2 is subject to AHR regulation. We conclude that AHR is a direct transcriptional regulator of human BCRP and provide an unprecedented role of AHR in cellular adaptive response and cytoprotection by up-regulating an important ATP-binding cassette efflux transporter.

Inhibition of multidrug resistance by adamantylgb3, a globotriaosylceramide analog.

Multidrug resistance (MDR) via the ABC drug transporter (ABCB1), P-glycoprotein (P-gp/MDR1) overexpression, is a major obstacle in cancer chemotherapy. Many inhibitors reverse MDR but, like cyclosporin A (CsA), have significant toxicities. MDR1 is also a translocase that flips glucosylceramide inside the Golgi to enhance neutral glycosphingolipid (GSL) synthesis. We observed partial MDR1/globotriaosylceramide (Gb3) cell surface co-localization, and GSL removal depleted cell surface MDR1. MDR1 may therefore interact with GSLs. AdamantylGb3, a water-soluble Gb3 mimic, but not other GSL analogs, reversed MDR1-MDCK cell drug resistance. Cell surface MDR1 was up-regulated 1 h after treatment with CsA or adaGb3, but at 72 h, cell surface expression was lost. Intracellular MDR1 accumulated throughout, suggesting long term defects in plasma membrane MDR1 trafficking. AdaGb3 or CsA rapidly reduced rhodamine 123 cellular efflux. MDR1 also mediates gastrointestinal epithelial drug efflux, restricting oral bioavailability. Vinblastine apical-to-basal transport in polarized human intestinal C2BBe1 cells was significantly increased when adaGb3 was added to both sides, or to the apical side only, comparable with verapamil, a standard MDR1 inhibitor. Disulfide cross-linking of mutant MDR1s showed no binding of adaGb3 to the MDR1 verapamil/cyclosporin-binding site between surface proximal helices of transmembrane segments (TM) 6 and TM7, but rather to an adjacent site nearer the center of TM6 and the TM7 extracellular face, i.e. close to the bilayer leaflet interface. Verotoxin-mediated Gb3 endocytosis also up-regulated total MDR1 and inhibited drug efflux. Thus, a functional interplay between membrane Gb3 and MDR1 provides a more physiologically based approach to MDR1 regulation to increase the bioavailability of chemotherapeutic drugs.

Carnitine/xenobiotics transporters in the human mammary gland epithelia, MCF12A.

The barrier function of the human mammary gland collapses if challenged with cationic drugs, causing their accumulation in milk. However, underlying molecular mechanisms are not well understood. To gain insight into the mechanism, we characterized transport of organic cations in the MCF12A human mammary gland epithelial cells, using carnitine and tetraethylammonium (TEA) as representative nutrient and xenobiotics probes, respectively. Our results show that the mammary gland cells express mRNA and proteins of human (h) novel organic cation transporters (OCTN) 1 and hOCTN2 (a Na+-dependent carnitine carrier with Na+-independent xenobiotics transport function), which belong to the solute carrier superfamily (SLC) of transporters. Other SLC OCTs such as hOCT1 and extraneuronal monoamine transporter (EMT)/hOCT3 are also expressed at mRNA levels, but hOCT2 was undetectable. We further showed mRNA expression of ATB0+ (an amino acid transporter with a Na+/Cl(-)-dependent carnitine transport activity), and Fly-like putative transporter 2/OCT6 (a splice variant of carnitine transporter 2: a testis-specific Na+-dependent carnitine transporter). TEA uptake was pH dependent. Carnitine uptake was dependent on Na+, and partly on Cl-, compatible with hOCTN2 and ATB0+ function. Modeling analyses predicted multiplicity of the uptake mechanisms with the high-affinity systems characterized by K(m) of 5.1 microM for carnitine and 1.6 mM for TEA, apparently similar to the reported hOCTN2 parameter for carnitine, and that of EMT/hOCT3 for TEA. Verapamil, cimetidine, carbamazepine, quinidine, and desipramine inhibited the carnitine uptake but required supratherapeutic concentrations, suggesting robustness of the carnitine uptake systems against xenobiotic challenge. Our findings suggest functional roles of a network of multiple SLC organic cation/nutrient transporters in human mammary gland drug transfer.