Molecular Mechanism of Taurocholate Transport by the Bile Salt Export Pump, an ABC Transporter Associated with Intrahepatic Cholestasis.

The bile salt export pump (BSEP/ABCB11) transports bile salts from hepatocytes into bile canaliculi. Its malfunction is associated with severe liver disease. One reason for functional impairment of BSEP is systemic administration of drugs, which as a side effect inhibit the transporter. Therefore, drug candidates are routinely screened for potential interaction with this transporter. Hence, understanding the functional biology of BSEP is of key importance. In this study, we engineered the transporter to dissect interdomain communication paths. We introduced mutations in noncanonical and in conserved residues of either of the two nucleotide binding domains and determined the effect on BSEP basal and substrate-stimulated ATPase activity as well as on taurocholate transport. Replacement of the noncanonical methionine residue M584 (Walker B sequence of nucleotide binding site 1) by glutamate imparted hydrolysis competency to this site. Importantly, this mutation was able to sustain 15% of wild-type transport activity, when the catalytic glutamate of the canonical nucleotide binding site 2 was mutated to glutamine. Kinetic modeling of experimental results for the ensuing M584E/E1244Q mutant suggests that a transfer of hydrolytic capacity from the canonical to the noncanonical nucleotide binding site results in loss of active and adoption of facilitative characteristics. This facilitative transport is ATP-gated. To the best of our knowledge, this result is unprecedented in ATP-binding cassette proteins with one noncanonical nucleotide binding site. Our study promotes an understanding of the domain interplay in BSEP as a basis for exploration of drug interactions with this transporter.

Virtual Screening of DrugBank Reveals Two Drugs as New BCRP Inhibitors.

The breast cancer resistance protein (BCRP) is an ABC transporter playing a crucial role in the pharmacokinetics of drugs. The early identification of substrates and inhibitors of this efflux transporter can help to prevent or foresee drug-drug interactions. In this work, we built a ligand-based in silico classification model to predict the inhibitory potential of drugs toward BCRP. The model was applied as a virtual screening technique to identify potential inhibitors among the small-molecules subset of DrugBank. Ten compounds were selected and tested for their capacity to inhibit mitoxantrone efflux in BCRP-expressing PLB985 cells. Results identified cisapride (IC50 = 0.4 µM) and roflumilast (IC50 = 0.9 µM) as two new BCRP inhibitors. The in silico strategy proved useful to prefilter potential drug-drug interaction perpetrators among a database of small molecules and can reduce the amount of compounds to test.

Subtle Structural Differences Trigger Inhibitory Activity of Propafenone Analogues at the Two Polyspecific ABC Transporters: P-Glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP).

The transmembrane ABC transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are widely recognized for their role in cancer multidrug resistance and absorption and distribution of compounds. Furthermore, they are linked to drug-drug interactions and toxicity. Nevertheless, due to their polyspecificity, a molecular understanding of the ligand-transporter interaction, which allows designing of both selective and dual inhibitors, is still in its infancy. This study comprises a combined approach of synthesis, in silico prediction, and in vitro testing to identify molecular features triggering transporter selectivity. Synthesis and testing of a series of 15 propafenone analogues with varied rigidity and basicity of substituents provide first trends for selective and dual inhibitors. Results indicate that both the flexibility of the substituent at the nitrogen atom, as well as the basicity of the nitrogen atom, trigger transporter selectivity. Furthermore, inhibitory activity of compounds at P-gp seems to be much more influenced by logP than those at BCRP. Exploiting these differences further should thus allow designing specific inhibitors for these two polyspecific ABC-transporters.

Flagging Drugs That Inhibit the Bile Salt Export Pump.

The bile salt export pump (BSEP) is an ABC-transporter expressed at the canalicular membrane of hepatocytes. Its physiological role is to expel bile salts into the canaliculi from where they drain into the bile duct. Inhibition of this transporter may lead to intrahepatic cholestasis. Predictive computational models of BSEP inhibition may allow for fast identification of potentially harmful compounds in large databases. This article presents a predictive in silico model based on physicochemical descriptors that is able to flag compounds as potential BSEP inhibitors. This model was built using a training set of 670 compounds with available BSEP inhibition potencies. It successfully predicted BSEP inhibition for two independent test sets and was in a further step used for a virtual screening experiment. After in vitro testing of selected candidates, a marketed drug, bromocriptin, was identified for the first time as BSEP inhibitor. This demonstrates the usefulness of the model to identify new BSEP inhibitors and therefore potential cholestasis perpetrators.

Transport of estradiol-17ß-glucuronide, estrone-3-sulfate and taurocholate across the endoplasmic reticulum membrane: evidence for different transport systems.

Important reactions of drug metabolism, including UGT mediated glucuronidation and steroidsulfatase mediated hydrolysis of sulfates, take place in the endoplasmic reticulum (ER) of hepatocytes. Consequently, UGT generated glucuronides, like estradiol-17ß-glucuronide, have to be translocated back into the cytoplasm to reach their site of excretion. Also steroidsulfatase substrates, including estrone-3-sulfate, have to cross the ER membrane to reach their site of hydrolysis. Based on their physicochemical properties such compounds are not favored for passive diffusion and therefore likely necessitate transport system(s) to cross the ER membrane in either direction. The current study aims to investigate the transport of taurocholate, estradiol-17ß-glucuronide, and estrone-3-sulfate in smooth (SER) and rough (RER) endoplasmic reticulum membrane vesicles isolated from Wistar and TR(-) rat liver. Time-dependent and bidirectional transport was demonstrated for taurocholate, showing higher uptake rates in SER than RER vesicles. For estradiol-17ß-glucuronide a fast time-dependent efflux with similar efficiencies from SER and RER but no clear protein-mediated uptake was shown, indicating an asymmetric transport system for this substrate. Estrone-3-sulfate uptake was time-dependent and higher in SER than in RER vesicles. Inhibition of steroidsulfatase mediated estrone-3-sulfate hydrolysis decreased estrone-3-sulfate uptake but had no effect on taurocholate or estradiol-17ß-glucuronide transport. Based on inhibition studies and transport characteristics, three different transport mechanisms are suggested to be involved in the transport of taurocholate, estrone-3-sulfate and estradiol-17ß-glucuronide across the ER membrane.

ATP-binding cassette transporters in liver.

The human ATP-binding cassette (ABC) superfamily consists of 48 members with 14 of them identified in normal human liver at the protein level. Most of the ABC members act as ATP dependent efflux transport systems. In the liver, ABC transporters are involved in diverse physiological processes including export of cholesterol, bile salts, and metabolic endproducts. Consequently, impaired ABC transporter function is involved in inherited diseases like sitosterolemia, hyperbilirubinemia, or cholestasis. Furthermore, altered expression of some of the hepatic ABCs have been associated with primary liver tumors. This review gives a short overview about the function of hepatic ABCs. Special focus is addressed on the localization and ontogenesis of ABC transporters in the human liver. In addition, their expression pattern in primary liver tumors is discussed.

Hepatocellular organic anion-transporting polypeptides (OATPs) and multidrug resistance-associated protein 2 (MRP2) are inhibited by silibinin.

Silibinin has been reported to be a promising compound for hepatitis C treatment of nonresponders to standard treatment. Although administered silibinin is well tolerated, increased serum bilirubin levels have been observed during high-dose i.v. silibinin therapy. The mechanism of silibinin-induced hyperbilirubinemia in humans, however, has not been identified so far. The aim of this study was to investigate the effect of silibinin on hepatocellular uptake and efflux transport systems for organic anions to elucidate the cause of silibinin-induced hyperbilirubinemia. Therefore, the effect of silibinin on transport activity of the hepatocellular uptake transporters organic anion-transporting polypeptides (OATPs) OATP1B1, OATP1B3, and OATP2B1, as well as Na(+)-taurocholate cotransporting polypeptide (NTCP) and of the efflux transporters multidrug resistance-associated protein 2 (MRP2) and bile-salt export pump (BSEP) was studied. The effect of silibinin on OATPs and NTCP function was studied in stable transfected Chinese hamster ovary cells using the radiolabeled model substrates estrone-3-sulfate and dehydroepiandrosteronesulfate for OATPs and taurocholate for NTCP. Interaction of silibinin with MRP2 and BSEP was measured in vesicles isolated from Sf21 or Sf9 insect cells expressing these transporters using either estradiol-17ß-glucuronide or taurocholate as substrates. OATP1B1, OATP1B3, and OATP2B1 were inhibited by silibinin, with OATP1B1 being inhibited by (a) complex mechanism(s). An inhibitory effect was also seen for MRP2. In contrast, the bile acid transporters NTCP and BSEP were not affected by silibinin. We concluded that silibinin-induced hyperbilirubinemia may be caused by an inhibition of the bilirubin-transporting OATPs and the efflux-transporter MRP2.

Tumor-specific expression of organic anion-transporting polypeptides: transporters as novel targets for cancer therapy.

Members of the organic anion transporter family (OATP) mediate the transmembrane uptake of clinical important drugs and hormones thereby affecting drug disposition and tissue penetration. Particularly OATP subfamily 1 is known to mediate the cellular uptake of anticancer drugs (e.g., methotrexate, derivatives of taxol and camptothecin, flavopiridol, and imatinib). Tissue-specific expression was shown for OATP1B1/OATP1B3 in liver, OATP4C1 in kidney, and OATP6A1 in testis, while other OATPs, for example, OATP4A1, are expressed in multiple cells and organs. Many different tumor entities show an altered expression of OATPs. OATP1B1/OATP1B3 are downregulated in liver tumors, but highly expressed in cancers in the gastrointestinal tract, breast, prostate, and lung. Similarly, testis-specific OATP6A1 is expressed in cancers in the lung, brain, and bladder. Due to their presence in various cancer tissues and their limited expression in normal tissues, OATP1B1, OATP1B3, and OATP6A1 could be a target for tumor immunotherapy. Otherwise, high levels of ubiquitous expressed OATP4A1 are found in colorectal cancers and their metastases. Therefore, this OATP might serve as biomarkers for these tumors. Expression of OATP is regulated by nuclear receptors, inflammatory cytokines, tissue factors, and also posttranslational modifications of the proteins. Through these processes, the distribution of the transporter in the tissue will be altered, and a shift from the plasma membrane to cytoplasmic compartments is possible. It will modify OATP uptake properties and, subsequently, change intracellular concentrations of drugs, hormones, and various other OATP substrates. Therefore, screening tumors for OATP expression before therapy should lead to an OATP-targeted therapy with higher efficacy and decreased side effects.

Gender- and dose-related effects of cyclosporin A on hepatic and bone metabolism.

Previous data have shown gender-related differences in the skeletal effects of the immunosuppressive drug cyclosporin A (CsA) in rats. To test the hypothesis that the gender-related skeletal effects of CsA are caused by gender-specific metabolism of this drug, we treated aged male and female sham-operated, gonadectomized (GX) as well as sex hormone-supplemented GX rats with 5 mg/kg CsA three times per week for 2 months, and analyzed the bone phenotype as well as the concentrations of CsA and its major metabolites AM1, AM1c, AM9, and AM4N in blood, urine, and liver tissue. CsA treatment induced high turnover osteopenia in males, but not females. Male rats showed several-fold higher CsA and CsA metabolite blood levels compared with females. Renal clearance data revealed that CsA undergoes selective tubular reabsorption in male, but not female rats. However, a mathematical modeling approach demonstrated that the higher CsA blood levels in males were almost exclusively caused by a 6-fold lower hepatic clearance rate compared with females. In addition, we subcutaneously treated female rats with up to 6-fold higher doses of CsA. Similar to males, high dose CsA induced high turnover osteopenia in female rats. Our data show that the gender-related differences in the skeletal effects of CsA are caused by a higher hepatic clearance rate for CsA in female compared to male rats, and not by a differential skeletal response to CsA. Moreover, our study indicates that CsA blood levels of ≤200 ng/ml measured by HPLC do not induce high turnover osteopenia in aged rats.

Expression of organic anion-transporting polypeptides 1B1 and 1B3 in ovarian cancer cells: relevance for paclitaxel transport.

PURPOSE: Ovarian cancer remains a deadly malignancy because most patients develop recurrent disease that is resistant to chemotherapy. Organic anion-transporting polypeptides (OATPs) mediate the uptake of clinically important drugs thereby effecting intracellular drug accumulation. In this study, we investigated whether OATPs may also contribute to paclitaxel transport in estrogen-responsive and estrogen-independent ovarian carcinoma cell lines and tumor tissue. METHODS: Expression of all 11 human OATPs in human ovarian cancer tissue samples and in the ovarian carcinoma cell lines OVCAR-3 and SK-OV-3 was investigated using real-time RT-PCR. Kinetic analysis of paclitaxel uptake was characterized in both cell lines and in OATP-transfected Xenopus laevis oocytes. Cytotoxicity of paclitaxel in OVCAR-3, SK-OV-3 and OATP1B1- and OATP1B3-transfected SK-OV-3 cells was performed using the CellTiter-Glo assay. RESULTS: OATP1B1 and OATP1B3 are active paclitaxel transporters in transfected X. laevis oocytes. Real-time RT-PCR analysis revealed expression of both OATPs in human ovarian cancer tissue specimens and in cancer cell lines. The higher mRNA levels for OATP1B1 and OATP1B3 found in SK-OV-3 cells correlated with higher initial uptake rates for paclitaxel. In addition, cytotoxicity studies with OATP1B1- and OATP1B3-transfected SK-OV-3 cells demonstrated lower IC(50) values compared to cells transfected with the empty vector. CONCLUSIONS: Our results revealed OATP1B1 and OATP1B3 as high-affinity paclitaxel transporters expressed in ovarian cancer cell lines and tumor tissues, suggesting a role for these polypeptides in the disposition of paclitaxel during therapy.

Melatonin receptors, melatonin metabolizing enzymes and cyclin D1 in human breast cancer.

BACKGROUND: Melatonin suppresses breast cancer cell proliferation by inhibiting the upregulation of estrogen-induced cyclin D1 via its G-protein-coupled receptor MT1. Additionally, melatonin stimulates the expression of the estrogen sulfotransferase, SULT1E1. However, metabolism of melatonin via 6-hydroxylation by CYP1A1/1A2 and subsequent sulfonation by SULT1A1/1A3 decreases its intracellular concentration. This could have a negative impact on its oncostatic action in breast cancer. PATIENTS AND METHODS: In this pilot study, we performed immunohistochemical (IHC) analysis of MT1 and cyclin D1 in breast cancer specimens from 33 patients. Also, we investigated the expression of CYP1A1/1A2, SULT1A1/1A3/1E1,and cyclin D1 in cancer (CANC) and adjacent non-cancer (NCANC) specimens from 10 representative breast cancer patients using quantitative real-time reverse transcription polymerase chain reaction. RESULTS: CYP1A1-mRNA-expression was found only in three CANC and in one NCANC. CYP1A2 mRNA was below the detection limit in all patients. SULT1A1 was observed only in two of the 10 CANC and one of the 10 NCANC specimens. But, all 10 CANC and NCANC samples showed high SULT1A3 levels. Cyclin D1 mRNA levels were found in all 10 CANC and NCANC specimens. Furthermore, IHC-staining of cyclin D1 was observed in 27 of 33 CANC and correlated positively with estrogen receptor positivity (p = 0.015). CONCLUSION: The low or even absent expression of CYP1A1 or CYP1A2 in breast cancer specimens suggested that melatonin might be involved in cell cycle arrest.

The analysis of organic anion transporting polypeptide (OATP) mRNA and protein patterns in primary and metastatic liver cancer.

Organic anion transporting polypeptides (OATP, SLCO genes) mediate the uptake of endobiotics and drugs. Thus, their expression levels and pattern could be of relevance for cancer therapy. This prompted us to investigate the expression of poorly characterized OATPs, namely OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in hepatic cancer of different origin. First, mRNA levels of all eleven OATPs were determined in paired (cancerous and adjacent non-cancerous) specimens from 43 patients with primary liver cancer (hepatocellular carcinoma, HCC; cholangiocellular carcinoma, CCC) and liver metastases from colon tumors (MLT). Real-time RT-PCR analysis revealed that all OATPs, except OATP1C1 and OATP6A1, are extensively expressed in nearly all samples. In contrast to downregulated OATP1B1, OATP1B3, OATP1A2 and OATP2B1 in cancerous vs. non-cancerous samples, an increase in OATP2A1, OATP3A1, OATP4A1 and OATP5A1 mRNA levels was seen in tumors (up to 40-fold for OATP5A1 in the MLT group). Therefore, OATP2A1, OATP3A1, OATP4A1 and OATP5A1 were further investigated by immunofluorescence microscopy on paraffin-embedded cancerous and non-cancerous sections (seven per group). OATP-derived immunoreactivity was observed in plasma membranes and cytosol of hepatic tumor cells, and additionally, in various cytokeratin 19 positive bile ducts. An increased percentage of immunoreactive cells and a higher staining intensity in cancerous vs. non-cancerous paraffin sections paralleled higher mRNA levels of OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in cancerous tissues of HCC, CCC and MLT patients. The extensive expression of OATP2A1, OATP3A1, OATP4A1 and OATP5A1 in hepatic tumors of different origin suggests that these transporters might be further exploited for the discovery of novel anticancer agents.

Organic anion transporting polypeptides (OATPs): regulation of expression and function.

Eleven members of the human organic anion transporter (OATP) family (grouped into six families) facilitate the Na(+)- independent transmembrane transport of various endo- and xenobiotics (bile acids, bilirubin, steroid hormone conjugates, thyroid hormones, prostaglandins, clinically used drugs, and toxins). OATPs are 12-transmembrane glycoproteins (643-722 amino acids) and contain many conserved structural features, for example, eleven cysteines in the large extracellular loop 5. They are important for proper transport, for which translocation of substrates through a central, positively-charged pore in a rocker-switch-type mechanism has been proposed. Although OATPs are expressed in various cells and tissues, some members show a more restricted pattern (well-studied OATP1B1/OATP1B3 in liver, OATP4C1 in kidney, and OATP6A1 in testis). In cancer, the distribution pattern is no longer maintained, and OATPs, like OATP1B3, become upregulated in malignant tissues (colon, breast, prostate). Studies in cell lines and animal models further revealed that the expression of OATPs is regulated in a cell- and tissue-specific way by cytokines and activation of nuclear receptors (LXR, FXR, PXR, CAR, HNF4). Also epigenetic mechanisms and postranslational modifications influence their expression and function. Therefore, changes in the expression of OATPs under pathological conditions will influence transport processes causing an altered accumulation of OATP substrates in cells of excretory organs (intestine, liver, kidney) and on various blood/organ barriers (such as brain, testis, placenta). For drugs, this may result in increased toxicity and adverse drug reactions. Therefore, it is important to improve the knowledge on the regulation and function of individual OATPs, and to apply it for therapeutic considerations.

Expression of sulfotransferases and sulfatases in human breast cancer: impact on resveratrol metabolism.

Resveratrol is a naturally occurring anticancer compound present in grapes and wine that undergoes pronounced metabolism in human intestine and liver. In order to determine whether resveratrol is also bio-transformed in human breast carcinoma, metabolism experiments were conducted in breast tumor and adjacent non-tumorous specimens from 13 patients. Resveratrol was metabolized in cytosolic tissue fractions to resveratrol-3-O-sulfate: the formation rates were up to 33.5-fold higher in cancer samples than in peritumoral tissue. Further quantitative real-time RT-PCR analysis revealed similar expression of sulfotransferases SULT1A2, 1A3, and 1E1 in the paired control and tumor tissues. Sulfotransferase SULT1A1 expression was below the detection limit in all samples. Interestingly, mRNA expression of steroid sulfatase STS, but not of arylsulfatases ARS-A and ARS-B, was significantly higher (p<0.0017) in non-malignant specimens than in tumor tissue samples, which might explain the higher resveratrol-3-O-sulfate concentrations in breast cancer specimens. Cellular localization of SULT1A3 and STS was also assessed by indirect immunofluorescence on paraffin-embedded sections from control and malignant breast tissue clearly showing a correlation of qRT-PCR data with protein expression of these two enzymes. Our data elucidate the metabolism of resveratrol in malignant and non-malignant breast tissue, which must be considered in humans after oral uptake of dietary resveratrol as a chemopreventive agent.

Different expression patterns of organic anion transporting polypeptides in osteosarcomas, bone metastases and aneurysmal bone cysts.

Organic anion transporting polypeptides (OATP) were identified as transmembrane transporters for various endo- and exogenous organic compounds (hormones, prostaglandins, anticancer drugs). OATP expression had been shown in different tissues, but not in bone tumors. Therefore, the expression pattern of all known eleven human OATPs was analyzed by quantitative RT-PCR in 21 human bone tumor specimens (osteosarcomas, bone metastases and benign aneurysmal bone cysts). Transcriptional expression of OATP1A2, 1C1, 2A1, 2B1, 3A1, 4A1, 4C1 and 5A1, but not of OATP1B1, 1B3 and 6A1 was observed in malignant and non-malignant tumor specimens at varying level. Importantly, OATP3A1, 4A1, 2B1 and 1C1 mRNA levels were significantly higher in aneurysmal bone cysts as compared to osteosarcomas. Elevated mRNA levels of OATP2A1, 1A2, and 4C1 in metastases from kidney cancer and of OATP5A1 in prostate cancer suggest that the OATP expression pattern in metastases is comparable to that of the primary tumors. Different to tissue, OATP expression in osteosarcoma cell lines HOS and MG-63, normal human osteoblast outgrowth cells (hOB) and bone marrow stromal cells (BMSC) is limited to OATP3A1 and OATP4A1. High OATP expression levels, particularly in benign bone tumors, suggest an important role of these transporters for providing hormones, their conjugates, prostaglandins and drugs in bone cells. Thereby, they may influence bone resorption and formation.

Chemical composition, olfactory evaluation and antioxidant effects of essential oil from Mentha canadensis.

The chemical composition of the essential oil from cornmint (Mentha canadensis L.) was analyzed by GC/FID and GC-MS. The main constituents were menthol (41.2%) and menthone (20.4%). It was established that cornmint oil had antiradical activity with respect to the DPPH and hydroxyl (OH*) radicals. The concentrations necessary for 50% neutralization of the respective radicals (IC50) were 365.0 microg/mL for DPPH and 0.3 microg/mL for OH*, which was indicative that the antioxidant activity in terms of OH* was higher than that of quercetin. Cornmint oil chelated the Fe3+ ions present in the solution. The oil demonstrated antioxidant activity in a linoleic acid emulsion model system, where at 0.1% concentration it inhibited the formation of conjugated dienes by 57.1% and the generation of secondary oxidized products of linoleic acid by 76.1%.

Antitumor activity of resveratrol and its sulfated metabolites against human breast cancer cells.

Resveratrol (3,4',5-trihydroxy- trans-stilbene) is a naturally occurring polyphenolic compound found in grapes, wine and medicinal plants with a variety of biological and pharmacological activities including pronounced anticancer properties. These effects are observed despite its extremely low bioavailability and rapid clearance from the circulation due to extensive sulfation and glucuronidation in the intestine and liver. In order to determine whether its metabolites demonstrate any cytotoxic properties, three major human sulfated conjugates of resveratrol were synthesized and their anticancer activity evaluated against three breast cancer cell lines (two hormone-dependent: MCF-7 and ZR-75-1; one hormone-independent: MDA-MB-231) and one immortalized breast epithelial cell line (MCF-10A). We found that, in contrast to resveratrol, all three sulfated metabolites were less potent against MCF-7, MDA-MB-231 and ZR-75-1 cells ( trans-resveratrol 3- O-sulfate < trans-resveratrol 4'- O-sulfate < trans-resveratrol 3- O-4'- O-disulfate) indicating that any conjugation of the phenolic groups with sulfuric acid strongly affecting the cytotoxicity. Interestingly, all sulfated metabolites were reduced about 10-fold, but showed nearly equal cytotoxicity towards nonmalignant MCF-10A breast cells (IC (50 s): 202-228 microM). In summary, in contrast to resveratrol its sulfated metabolites showed poor cytotoxicity in human malignant and nonmalignant breast cancer cell lines. However, the in vitro activity of the metabolites may not necessarily reflect their in vivo function, given the fact that the ubiquitously existing human sulfatases could convert the metabolites back to resveratrol in humans.

Altered expression of organic anion transporter polypeptide (OATP) genes in human breast carcinoma.

Organic anion transporter polypeptides (OATPs) mediate the transmembrane uptake of endogenous compounds and clinically important drugs in various tissues thereby effecting drug disposition and tissue penetration. OATPs have also been identified in gastric, pancreatic and colon carcinomas but little is known about their expression in breast carcinoma. We therefore analyzed the expression pattern of all 11 known OATPs in three breast cancer cell lines (MCF-7, ZR-75-1, MDA-MB-231) and one immortalized breast epithelial cell line (MCF-10A) using quantitative real-time RT-PCR. Transcripts of 7/11 OATP genes with heterogeneity in their expression profile were detected in control and/or cancer cell lines. Of these seven OATPs, five were also expressed in breast tumor and adjacent non-tumorous specimens from 13 patients. OATP2B1, not found in the analyzed cell lines, was verified in the tissue samples. Interestingly, mRNA expression of OATP2B1, OPATP3A1 and OATP4A1 was significantly higher (p < 0.022) in non-malignant specimens as compared to tumor tissue samples.