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Expression and localization of the uptake transporters OATP2B1, OATP3A1 and OATP5A1 in non-malignant and malignant breast tissue

Expression and localization of the uptake transporters OATP2B1, OATP3A1 and OATP5A1 in... Resea RCh pape R Cancer Biology & Therapy 11:6, 584-591; March 15, 2011; © 2011 Landes Bioscience Expression and localization of the uptake transporters OATP2B1, OATP3A1 and OATP5A1 in non-malignant and malignant breast tissue 1 2 2 3 3 2 2 Juergen Kindla, Tilman T. Rau, Rudolf Jung, peter a . Fasching, Reiner s trick, Robert s toehr, a rndt h artmann, 1 1, Martin F. Fromm and Jörg König * Institute of experimental and Clinical pharmacology and Toxicology; Clinical pharmacology and Clinical Toxicology; 2 3 Friedrich-a lexander-University erlangen-Nuremberg; erlangen, Germany; and Institute of pathology; Department of Gynecology and Obstetrics; University h ospital erlangen; erlangen, Germany Key words: OATP, breast cancer, expression, localization, transport Abbreviations: OATP, organic anion transporting polypeptide; SLC, solute carrier superfamily; PCR, polymerase chain reaction; TBS, tris-buffered saline; E3S, estrone-3-sulfate; DHEAS, dehydroepiandrosterone-3-sulfate Organic anion transporting polypeptides (Oa Tps, gene family SLCO/SLC21) mediate the uptake of multiple endogenous substances such as estrogens and estrogen metabolites and of several widely prescribed drugs (e.g., statins, antibiotics and anticancer agents) into cells. s ince several anticancer agents have been identified as substrates for O aTps, these transporters may also have an impact on cancer treatment. expression of Oa Tps has been identified in colon, pancreatic and gastric carcinomas but to date little is known about the expression and localization of Oa Tp family members in non- malignant breast tissue and breast cancer. We therefore analyzed the mRNa expression of all human OaTp family members and further evaluated the mRNa amount of the highly-expressed Oa Tps Oa Tp2B1, Oa Tp3a 1 and Oa Tp5a 1 in 10 paired samples of normal breast tissue and ©2011 Landes Bioscience. breast cancer. Furthermore, the tissue-specific localization of these O a Tps was investigated. The results demonstrated that the mRNa expression of Oa Tps in normal and malignant breast tissue shows a high interindividual variability and that no significant differences in the mRN a amount between normal and malignant tissue could be detected. Further- Do not distribute. more, we localized Oa Tp3a1 and Oa Tp5a1 to the plasma membrane of epithelial cells of the lactiferous ducts in normal breast tissue. In breast cancer, both Oa Tps are highly expressed in the plasma membrane and in the cytoplasm. s ince estrogen and estrogen metabolites as well as anticancer agents are substrates for Oa Tps these results indicate the pos- sibility of OaTp-mediated uptake of hormones during breast cancer development and an impact of certain OaTps on chemotherapeutic cancer treatment. 5,6 by OATP1B1 and OATP1B3, Introduction whereas docetaxel and paclitaxel have been identified as substrates for OATP1B3, and gimatecan Members of the human organic anion transporting polypeptide was transported by OATP1B1. In addition to OATP1B1 and family (OATPs; SLC gene family SLC21/SLCO) are important OATP1B3 methotrexate is also transported by OATP4C1. uptake transporters expressed in various organs such as liver, Whereas OATP1B1 and OATP1B3 are predominantly 1,2 kidney, placenta and in the blood-brain barrier. Today, eleven expressed in human liver, other family members such as OATP2B1 human OATP family members have been identified and grouped and OATP4A1 seem to be ubiquitously expressed (for detailed into six different subfamilies (OATP1–OATP6). They mediate description of the tissue expression of human OATP family mem- the sodium-independent uptake of a broad range of amphipatic bers see the review in reference 2). In addition to the expression organic anions from the extracellular environment into cells. in normal, non-malignant tissues, OATPs are present in several Substrates for human OATPs include endogenous substances tumorous tissues and cancer cell lines. OATP1B1 and OATP1B3 such as hormones or hormone metabolites, bile salts and xeno- have been detected in hepatocellular carcinomas, whereas no biotics including several widely prescribed drugs such as HMG- expression of these transporters was found in liver metastases of CoA-reductase inhibitors (statins), antihypertensive drugs and colorectal and pancreatic adenocarcinoma. OATP1B3 seems 1,3,4 11 antibiotics. Furthermore, recent studies have shown that to be overexpressed in colorectal adenocarcinomas. In human several anticancer agents are substrates for some human OATP glioma specimens mRNA expression of OATP1A2, OATP1C1, family members. Atrasentan and methotrexate are transported OATP2B1 and OATP4A1 has been detected. *Correspondence to: Jörg König; Email: joerg.koenig@pharmakologie.med.uni-erlangen.de Submitted: 09/29/10; Revised: 12/15/10; Accepted: 12/19/10 DOI: 10.4161/cbt.11.6.14533 584 Cancer Biology & Therapy Volume 11 Issue 6 Resea RCh pape R Resea RCh pape R Controversial data have been published for the expression demonstrating that only OATP2B1, OATP3A1 and OATP5A1 of OATPs in normal human mammary epithelial cells and mRNA were expressed in adequate amounts when compared to breast cancer. In mammary epithelia cells mRNA expression of the expression of the housekeeping gene b-actin. Primer pairs OATP1A2, OATP2B1, OATP3A1 and OATP4A1 was detected and amplified fragment length are shown in Table 1 . Whereas and the expression of OATP1A2, OATP3A1 and OATP4A1 was these three OATPs were expressed with percentage values (related 14,15 also found in T-47D and MCF-7 breast cancer cell lines. to b-actin) of more than 0.1%, all other OATP family members Interestingly, Pizzagalli and coworkers confirmed the mRNA have expression values below 0.1% b-actin expression suggesting expression of OATP3A1 and OATP4A1, but could not detect a that only OATP2B1, OATP3A1 and OATP5A1 are of significant significant OATP1A2 mRNA expression in normal breast tis- interest in non-malignant and malignant breast tissue. 16 17 sue. In human breast cancer, one study found an overexpres- Next, we investigated the mRNA expression of these three sion of OATP1A2 mRNA compared to corresponding normal uptake transporters in ten normal mammary tissue samples and mammary tissue, whereas no OATP1A2 mR NA expression could in matched samples from the corresponding breast cancer from be detected in a study analyzing the mRNA expression of all the same patient. Clinical characteristics of patients are summa- OATP family members in 13 breast cancer samples. This study rized in Table 2. OATP2B1, OATP3A1 and OATP5A1 mRNA demonstrated the mRNA expression of OATP2A1, OATP2B1, were highly expressed in normal mammary tissue and in matched OATP3A1, OATP4A1, OATP4C1 and OATP5A1, whereas breast cancer with average expression values of 1.4% of b-actin expression of OATP1A2, OATP1B1, OATP1B3, OATP1C1 and mRNA expression for OATP2B1 [95% CI: 0.8–2.0], 3.9% of OATP6A1 was below the detection limit. A recent study by b-actin mRNA expression for OATP3A1 [95% CI: 2.0–5.7%] Maeda et al. analyzed the expression of OATP family mem- and 12.6% of b-actin mRNA expression for OATP5A1 [95% bers in two subclones of MCF-7 breast cancer cells with dif- CI: 5.3–20.0%]. No significant differences between normal ferent estrone-3-sulfate (E3S) uptake activities, demonstrating mammary tissue and tumor tissue were observed (Fig. 1). that among several candidate transporters OATP1B3 might be Localization of OATP2B1, OATP3A1 and OATP5A1 in responsible for the uptake of E3S into MCF-7 cells. Taking these normal human mammary tissue and breast cancer tissue sam- data together it is evident that the results of the mRNA expres- ples. The localization of OATP2B1, OATP3A1 and OATP5A1 sion data of OATP family members in breast cancer tissue or was analyzed using immunohistochemical (Fig. 2) and immu- breast cancer cell line© s are co2 ntro0 vers1 ial a1 nd h aL ve ta o be sn tudd ied ie n s noflu oB rescei nco e ans alyc ses (ie Fig.n 3). Sc pe ecifici. ty of the respective more detail. Moreover, only the protein expression and localiza- antisera used for these analyses has been verified in immunoblot tion of OATP2B1 has been analyzed so far with a lack of protein experiments using homogenates of stably transfected cell lines data for other OATP family members. expressing OATP family members (data not shown). Do not distribute. Based on these expression data and due to the importance of OATP2B1, OATP3A1 and OATP5A1 were detected by OATPs for the cellular uptake of drugs including chemothera- immunohistochemistry in lobules and ducts and in capillary peutic agents it became evident that the detailed knowledge on endothelium in healthy mammary tissue samples and were found the expression of OATP family members in breast cancer tissue to be localized in the cytoplasm and in the plasma membrane of and even more on the tissue specific localization of the expressed malignant cells (Fig. 2). In accordance with the data from immu- proteins can be important for cancer treatment. Furthermore, nohistochemical analyses, immunofluorescence analyses demon - estrogens and estrogen metabolites are substrates of some OATPs strated (Fig. 3) that OATP2B1, OATP3A1 and OATP5A1 are and since these hormones are important for breast cancer pro- localized in the plasma membrane of epithelial cells of lactiferous gression, presence of OATP uptake transporters in plasma mem- ducts. In corresponding tumorous cells the three proteins were branes may be a prerequisite for the intracellular action of these localized in the plasma membrane and to a major extend in the signalling molecules. Therefore, the aim of this study was to cytoplasm. analyze expression of OATP family members in paired samples of normal mammary tissue and corresponding breast cancer Discussion tissue. Furthermore, the cell specific localization of OATP2B1, OATP3A1 and OATP5A1, all highly expressed in mammary epi- Transporter-mediated uptake from the extracellular space into cells thelial cells and breast cancer was investigated by immunohisto- has been recognized as a fundamental step for endogenous pro- chemistry and immunofluorescence analysis. cesses and for drug disposition. Therefore, the knowledge on the tissue-specic e fi xpression of uptake transporters is important for the Results understanding of physiological and pathophysiological processes as well as for drug therapy. In this manuscript we investigated the Expression of mRNA of OATP family members in normal mRNA expression of all human OATP family members and ana- human mammary tissue and breast cancer tissue samples. First, lyzed the localization of the highly expressed OATPs OATP2B1, the mRNA expression of the human OATP family members OATP3A1 and OATP5A1 both in normal mammary tissue and except of the prostaglandin transporter OATP2A1 (OATP1A2, corresponding breast cancer tissue. The mRNA expression analy- OATP1B1, OATP1B3, OATP1C1, OATP2B1, OATP3A1, ses demonstrated that only OATP2B1, OATP3A1 and OATP5A1 OATP4A1, OATP4C1, OATP5A1 and OATP6A1) in five paired are expressed in significant amounts in normal mammary tissue samples of normal breast tissue and breast cancer was investigated samples and that no differences in the amount of mRNA was www.landesbioscience.com Cancer Biology & Therapy 585 Table 1. s equence of primers used in quantitative real-time pCR Forward primer Reverse primer Fragment length Oa Tp1a 2 5'-TGG CTG CCa Ta C CTG Ga T a Ta T-3' 5'-aa T Ta a GT T GTa Ca G Ca T GT T CTC-3' 492 bp Oa Tp1B1 5'-TGC a CT TGG a GG Ca C CTC a C-3' 5'-CT T Ca T CCa TGa Ca C T TC Ca T T T-3' 359 bp Oa Tp1B3 5'-TCa Ta a a CT CT T TGT TCT CTG Ca a -3' 5'-GT T GGC a GC a GC a T T GTC T TG-3' 482 bp Oa Tp1C1 5'-CCT GGa Ta C a Ta T Ta CT T CTG a G-3' 5'-Ca T GT T TCT aaa GT T Ga G T T T CCT-3' 436 bp Oa Tp2B1 5'-TGC TCa TCC Ta a Ga G Ga G TGa a -3' 5'-CCC aa G a Ca GCT Ca C a CT CG-3' 391 bp Oa Tp3a 1 5'-GT T GGG CT T Ca T CCC TCC a C-3' 5'-T Ta GTC a CT a Ta a aa CGG a CT CC-3' 391 bp Oa Tp4a 1 5'-Ga G a CT GTa GCT GTa TCC CTC-3' 5'-GCG GTG GTC a Ga CGC TGC T-3' 531 bp Oa Tp4C1 5'-CTC CTa Ta a CTG TGT CTa TCC T-3' 5'-CCC a T T TCa CCC T TC T T T Ta C T-3' 394 bp Oa Tp5a 1 5'-Ca a Ca G Ga a TGT GGT GTG Ca G-3' 5'-a GC T TC a GG a GG GCG GCT C-3' 402 bp Oa Tp6a 1 5'-Ga T GCa a a G TGC Ta T aa G T Ta -3' 5'-TCC a GT Ta C aa G TCa GT T TCT T-3' 451 bp Table 2. Tumor characteristics of tissue samples Primary Lymph node Metastasis Estrogen Progesterone Patient no. HER-2 status Histological type a a a tumor stage status status receptor status receptor status 1 pT1b N0 M0 - - - n.a. 2 pT1c N0 M0 - - - n.a 3 pT2 N0 M0 - - - ductal 4 pT4b N1 M1 + + - lobular 5 pT2 N1 M0 + + - lobular 6 pT1c N0 M0 + + - ductal ©2011 Landes Bioscience. 7 pT1c N0 M0 + + - ductal 8 pT2 N0 M0 + + - lobular 9 pT1c N1 M0 + - + ductal Do not distribute. 10 pT2 N0 M0 + + - ductal a th a ccording TNM classification, 6 edition, 2003. n.a., not available. observed between normal mammary tissue and corresponding reported recently that they have obtained two subclones of MCF-7 breast cancer tissue. The expression data are largely in line with cells with different estrone-3-sulfate uptake activities and differ- previously published work, in which OATP2B1, OATP3A1 and entially expressed transporter genes supporting the hypothesis of OATP5A1 mRNA was reported to be highly expressed in malig- different cell sublines. nant and non-malignant breast tissue samples. Furthermore, Since the mRNA expression varies between tissue samples mRNA expression of OATP1A2, OATP4A1 and OATP4C1 has and differs to the expression in cancer cells lines we subsequently 17,18 also been detected in some breast cancer tissue samples. This analyzed the localization of the OATP proteins in mammary could be confirmed in our study, but the expression of these OATPs tissue and corresponding cancer tissue. Therefore, we used was very low compared to the expression of OATP2B1, OATP3A1 immunohistochemical (Fig. 2) and immunofluorescence analy - and OATP5A1. Interestingly, as shown in Figure 1, the interin- ses (Fig. 3) to study the localization of the highly expressed dividual variability of the mRNA expression is very high (95% OATPs OATP2B1, OATP3A1 and OATP5A1 in normal breast CI for OATP5A1: 5.3–20.0% b-actin expression) suggesting that tissue and corresponding malignant tissue samples. For the first interindividual differences in gene regulation exists. A discrepancy time we could demonstrate that OATP3A1 and OATP5A1 are could be observed comparing the mRNA expression data in non- localized in the plasma membrane of epithelial cells of the lactifer- malignant or malignant breast cancer tissue with the expression ous ducts in non-malignant breast tissue and also in the plasma in human breast cancer derived cancer cells (e.g., MCF-7 cells). membrane and to a major extend cytoplasmatic in tumorous cells. Expression of OATP1B3 has been detected in MCF-7 cells, Furthermore, we could confirm the previously published local - whereas no expression of OATP1B3 mRNA has been observed ization of OATP2B1, in the same tissue compartments. These in tissue samples. Furthermore, Wlcek et al. have not detected results are of interest for cancer treatment since anticancer agents OATP1B3 mRNA in MCF-7 cells or in non-malignant MCF-10A used in the treatment of breast cancer have been identie fi d as sub - breast cells suggesting that maybe different sublines of MCF-7 cells strates for OATP family members. Paclitaxel and docetaxel have are present in different laboratories or that cell culture conditions been identie fi d being a substrate for OATP1B3, and it has been may be responsible for altered mRNA expression. Maeda et al. demonstrated that OATP1B3 determines methotrexate sensitivity 586 Cancer Biology & Therapy Volume 11 Issue 6 23 23 28 OATP1B1, OATP1B3, and OATP2B1. These previous results demonstrated that both highly expressed OATP family members OATP2B1 and OATP3A1 could be important for the hormonal regulation of breast cells and may also be involved in the progression of breast cancer due to estrogens. Since hormone receptors are located intracellularly, the uptake of hormones and hormone metabolites is a prerequisite for their subsequent regu- latory properties. Therefore, the association between genetic variations in transporter genes encoding uptake transporters for estrogen and estrogen metabolites with breast cancer risk has been studied recently by Justenhoven et al. Interestingly, they demon- strated that none of the frequent polymorphisms in genes encod- ing OATP1A2, OATP1B1, OATP1B3 and OATP2B1 showed an association with the risk for breast cancer suggesting that other OATPs are important for the uptake of these hormones in normal breast tissue and breast cancer. So far no substrates have been iden- tie fi d for OATP5A1 demonstrating the need of further research regarding the role of this uptake transporter for the transport of endogenous substances or drugs. Taken together we have analyzed the mRNA expression of all human OATP family members in non-malignant and malig- nant breast tissue samples and have compared the mRNA expres- sion of the highly expressed OATPs OATP2B1, OATP3A1 and OATP5A1 between normal mammary tissue and matched samples from corresponding tumorous breast tissue samples. Furthermore, ©2011 Landes the t B issue-i spo ecic l fis ocac lizati ioe n on f thec se te hree u. ptake transporters has been analyzed in normal mammary tissue and correspond- ing breast cancer tissue samples. We could demonstrate that these three uptake transporters are localized in the plasma membrane Do not distribute. of epithelial cells of the lactiferous ducts and in addition in the plasma membrane and to a greater extend intracellularly in breast cancer tissue cells. Since estrogen and estrogen metabolites are sub- strates of OATP2B1 and OATP3A1 these results are important for further studies on the molecular action of these hormones and may be relevant for cancer treatment using anticancer agents being substrates for these uptake transporters. Whether selective estro- gen receptor modulators (SERMs; e.g., Tamoxifen), which behave Figure 1. OATP2B1, OATP3A1 and OATP5A1 expression in normal hu- as estrogen receptor antagonists, are also substrates of the OATP man mammary and breast cancer tissue. Quantitative real-time pCR family members expressed in breast cancer has to be elucidated in demonstrated that OATP2B1, OATP3A1 and OATP5A1 mRNa are highly the future. expressed in normal mammary tissue and in corresponding breast can- cer with average expression values of 1.4% of b-actin for OATP2B1 [95% CI: 0.8–2.0%] (a ), 3.9% of b-actin for OATP3A1 [95% CI: 2.0–5.7%] (B) and Materials and Methods 12.6% of b-actin for OATP5A1 [95% CI: 5.3–20.0%] (C). expression levels of all other human Oa Tp family members were significantly lower. No Antibodies and reagents. The polyclonal antibodies AVE and differences between normal mammary tissue and breast cancer were SSS were raised in rabbits against the COOH-terminal sequences observed, p > 0.05. of OATP2B1 (AVE QQL LVS GPG KKP EDS RV A A 690 –709) and OATP5A1 (SSS ADP GLE ESP A AL EPP S A A 830–848), in gastrointestinal cancers. Moreover, several estrogens and estro- respectively. The polyclonal antibody RTK was raised in guinea gen metabolites have been identie fi d as substrates for OATP family pig against the COOH-terminal sequence of OATP3A1 (RTK members and since the mammary gland is an estrogen-responsive FIY NLE DHE WCE NME SVL A A 690–710). Antibodies SSS tissue, uptake of these hormones are important for lobular devel- and RTK were a kind gift from Prof. Dr. D. Keppler (German opment, regulation of epithelia cell growth or steroid hormone Cancer Research Center, Heidelberg, Germany). Cy3-conjugated 20,21 metabolizing enzymes. Estrone-3-sulfate has been identie fi d as AffiniPure goat anti-rabbit IgG and Cy3-conjugated AffiniPure 22 23 24 25 substrate for OATP1A2, OATP1B1, OATP1B3, OATP1C1, donkey anti-guinea pig IgG were obtained from Dianova, cat# 26 26 26 OATP2B1, OATP3A1 and OATP4A1, whereas dehydroepi- 111-165-144 and cat# 706-165-148, respectively. HRP-labeled androsterone-3-sulfate (DHEAS) is a substrate for OATP1A2, Polymer was from EnVisionTM, Dako, cat# K6061. All other www.landesbioscience.com Cancer Biology & Therapy 587 ©2011 Landes Bioscience. Do not distribute. Figure 2. Immunohistochemistry of Oa Tp2B1 (a and D), Oa Tp3a 1 (B and e) and Oa Tp5a 1 (C and F). Immunohistochemical experiments were per- formed using an antiserum directed against human Oa Tp2B1 (a Ve), an antiserum directed against human Oa Tp3a 1 (RTK) and an antiserum directed against human Oa Tp5a 1 (s ss ). Membranous and cytoplasmatic staining was regarded as positive. In normal tissue Oa Tp2B1, Oa Tp3a 1 and Oa Tp5a 1 were detected in lobules (arrows) and ducts (arrow-heads). a ll three targets showed neither in quantity nor in intensity differences between normal tissue (a –C) and breast cancer (D–F). Bars correspond to 100 μm. Figure 3 (See opposite page). Localization of Oa Tp2B1, Oa Tp3a 1 and Oa Tp5a 1 in human normal breast tissue and breast cancer. Oa Tp2B1 is localized in the plasma membrane of epithelial cells of the lactiferous duct and in capillary endothelium. Oa Tp3a 1 and Oa Tp5a 1 are localized in the plasma membrane of epithelial cells of the lactiferous duct. In breast cancer Oa Tp2B1, Oa Tp3a 1 and Oa Tp5a 1 are localized in the plasma membrane and to a major extend in the cytoplasm. 588 Cancer Biology & Therapy Volume 11 Issue 6 ©2011 Landes Bioscience. Do not distribute. Figure 3. For figure legend, see page 588. www.landesbioscience.com Cancer Biology & Therapy 589 chemicals and reagents, unless stated otherwise, were obtained with 2% bovine serum albumin and incubated with a polyclonal from Carl Roth GmbH + Co. KG, Karlsruhe Germany and were rabbit antiserum directed against human OATP2B1 (AVE), a of the highest grade available. polyclonal guinea pig anti-human OATP3A1 (RTK) or a poly- Tissue samples. Human normal and tumorous breast tissue clonal rabbit anti-human OATP5A1 antibody (SSS). Whereas was obtained from patients with incident invasive breast cancer the AVE antiserum is able to detect OATP2B1 proteins encoded undergoing routine primary surgery between 2002–2006 at the by the reference sequence and by known splice variants, the RTK Department of Gynaecology and Obstetrics, Erlangen University antiserum detects only the OATP3A1 protein encoded by the ref- Hospital, Erlangen, Germany. Use of the tissues was approved by erence sequence. Cy3-conjugated AffiniPure donkey anti-guinea the Ethics Committee at the Friedrich-Alexander-University of pig and goat anti-rabbit IgG were used as secondary antibodies. Erlangen-Nuremberg. All patients gave a written informed con- Nuclei were counterstained with SYTOX Green dye (Invitrogen sent. Tissues were collected at the Department of Gynecology and GmbH, cat# S7020). Samples were fixed with cover slips and an Obstetrics (Erlangen, Germany) and classie fi d by the Institute of aqueous mounting medium (Thermo Scientific, cat# 9990402). Pathology, Friedrich-Alexander-University Erlangen-Nuremberg, A confocal laser scanning microscope Axiovert 100M (Carl Erlangen, Germany. Paraffin-embedded tissue samples were Zeiss GmbH, Jena, Germany) was used to visualize the fluores - obtained by the Institute of Pathology (Erlangen, Germany). cence. Images were further processed using the Zeiss LSM Image RNA extraction. Total RNA was isolated from frozen tissue Browser version 4.0.0.157. samples using RNeasy Mini Kit (Quiagen GmbH, cat# 74104) Immunohistochemical analysis. The paraffin sections were according to the manufacturer’s instructions. Concentration and deparaffinized with xylene and rehydrated with graded ethanol. purity of RNA samples was determined by UV absorbance. Slides were subjected to a 5 min heating at 120°C in Tris-EDTA- Quantitative real-time PCR. Messenger-RNA was reverse Buffer pH 8.5 in a pressure cooker. Endogenous peroxidase activity transcribed to cDNA using oligo(dT ) primers and Affinity was quenched by a five min incubation with Peroxidase-Blocking TM Script Multiple Temperature Reverse Transcriptase (Stratagene, solution, Dako REAL (Dako, cat# S2023). Afterwards the sec- cat# 200436). The amount of mRNA was measured by tions were incubated with the polyclonal rabbit antiserum directed quantitative real-time PCR using the LightCycler 2 System against human OATP2B1 (AVE), the purie fi d polyclonal guinea (Roche Diagnostics GmbH) and normalized to the amount pig antiserum directed against human OATP3A1 (RTK) or the of the mRNA of th© e hous2 ekee0 ping g1 en1 e b -L actia n. Tn he ed xpere i-s pol ycB lonal rio abbit as nc tiseri um den irectc ed ae gain. st human OATP5A1 ments were performed using the LightCycler FastStart DNA (SSS) for 30 min. Slides were subsequently incubated with the Plus TM Master SYBR Green I reagents (Roche, cat# 03515885001) HR P-labeled Polymer EnVision + Dual Link (Dako, cat# K4061) and the primers pairs described in Table 1. All primer pairs for 30 min. Antibody binding was visualized using 3,3'-diamino- Do not distribute. are specific for detecting the respective OATP family member. benzidine in chromogen solution, Liquid DAB + Substrate (Dako, For OATP2B1, the primer pair detects all described splice vari- cat# K3467). Tissue sections were counterstained with hematoxy- ants whereas for OATP3A1 only the cDNA of the reference lin (Merck) to visualize tissue structures. The specic fi ity of immu - sequence (NM_013272.3) was detected. Amplification of PCR noreactive signals for OATP2B1, OATP3A1 and OATP5A1 was fragments was started by a denaturation step of 10 min at 95°C, verie fi d by negative controls, which were incubated without the followed by 45 cycles of denaturation at 95°C for 10 s, anneal- respective primary antiserum. ing at 64°C and elongation for 30 s at 72°C. At the end of DNA Statistical analysis. The expression of OATP2B1, OATP3A1 amplification, analyses of melting curves and agarose gel elec - and OATP5A1 mRNA in healthy mammary tissue and corre- trophoresis analyses were performed. sponding cancer tissue samples was analyzed using Mann-Whitney Immunoufl orescence analysis. Cryosections of healthy test. A p value < 0.05 was considered statistically signic fi ant. mammary tissue and tumorous tissue (5 μm thickness) were Acknowledgements prepared using the 2800 Frigocut N (Reichert Jung, now Leica Microsystems Nussloch GmbH). Tissues were fixed in ice-cold The authors’ work is supported by a grant from the Deutsche 70% methanol for 10 min. Subsequently, samples were permea- Krebshilfe (107854) as well as by a grant from the Deutsche bilized using TBS/Triton for 10 min. The tissues were blocked Forschungsgemeinschaft (DFG Ko 2120/1–3). 590 Cancer Biology & Therapy Volume 11 Issue 6 11. Lee W, Belkhiri A, Lockhart AC, Merchant N, Glaeser 21. Katzenellenbogen BS, Montano MM, Ediger TR, Sun References H, Harris EI, et al. Overexpression of OATP1B3 J, Ekena K, Lazennec G, et al. Estrogen receptors: 1. König J, Seithel A, Gradhand U, Fromm MF. confers apoptotic resistance in colon cancer. Cancer selective ligands, partners and distinctive pharmacol- Pharmacogenomics of human OATP transport- Res 2008; 68:10315-23; PMID: 19074900; DOI: ogy. Recent Prog Horm Res 2000; 55:163-93; PMID: ers. 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Lab Invest 2003; 83:527-38; PMID: 12695556. www.landesbioscience.com Cancer Biology & Therapy 591 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cancer Biology & Therapy Taylor & Francis

Expression and localization of the uptake transporters OATP2B1, OATP3A1 and OATP5A1 in non-malignant and malignant breast tissue

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Copyright © 2011 Landes Bioscience
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10.4161/cbt.11.6.14533
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Abstract

Resea RCh pape R Cancer Biology & Therapy 11:6, 584-591; March 15, 2011; © 2011 Landes Bioscience Expression and localization of the uptake transporters OATP2B1, OATP3A1 and OATP5A1 in non-malignant and malignant breast tissue 1 2 2 3 3 2 2 Juergen Kindla, Tilman T. Rau, Rudolf Jung, peter a . Fasching, Reiner s trick, Robert s toehr, a rndt h artmann, 1 1, Martin F. Fromm and Jörg König * Institute of experimental and Clinical pharmacology and Toxicology; Clinical pharmacology and Clinical Toxicology; 2 3 Friedrich-a lexander-University erlangen-Nuremberg; erlangen, Germany; and Institute of pathology; Department of Gynecology and Obstetrics; University h ospital erlangen; erlangen, Germany Key words: OATP, breast cancer, expression, localization, transport Abbreviations: OATP, organic anion transporting polypeptide; SLC, solute carrier superfamily; PCR, polymerase chain reaction; TBS, tris-buffered saline; E3S, estrone-3-sulfate; DHEAS, dehydroepiandrosterone-3-sulfate Organic anion transporting polypeptides (Oa Tps, gene family SLCO/SLC21) mediate the uptake of multiple endogenous substances such as estrogens and estrogen metabolites and of several widely prescribed drugs (e.g., statins, antibiotics and anticancer agents) into cells. s ince several anticancer agents have been identified as substrates for O aTps, these transporters may also have an impact on cancer treatment. expression of Oa Tps has been identified in colon, pancreatic and gastric carcinomas but to date little is known about the expression and localization of Oa Tp family members in non- malignant breast tissue and breast cancer. We therefore analyzed the mRNa expression of all human OaTp family members and further evaluated the mRNa amount of the highly-expressed Oa Tps Oa Tp2B1, Oa Tp3a 1 and Oa Tp5a 1 in 10 paired samples of normal breast tissue and ©2011 Landes Bioscience. breast cancer. Furthermore, the tissue-specific localization of these O a Tps was investigated. The results demonstrated that the mRNa expression of Oa Tps in normal and malignant breast tissue shows a high interindividual variability and that no significant differences in the mRN a amount between normal and malignant tissue could be detected. Further- Do not distribute. more, we localized Oa Tp3a1 and Oa Tp5a1 to the plasma membrane of epithelial cells of the lactiferous ducts in normal breast tissue. In breast cancer, both Oa Tps are highly expressed in the plasma membrane and in the cytoplasm. s ince estrogen and estrogen metabolites as well as anticancer agents are substrates for Oa Tps these results indicate the pos- sibility of OaTp-mediated uptake of hormones during breast cancer development and an impact of certain OaTps on chemotherapeutic cancer treatment. 5,6 by OATP1B1 and OATP1B3, Introduction whereas docetaxel and paclitaxel have been identified as substrates for OATP1B3, and gimatecan Members of the human organic anion transporting polypeptide was transported by OATP1B1. In addition to OATP1B1 and family (OATPs; SLC gene family SLC21/SLCO) are important OATP1B3 methotrexate is also transported by OATP4C1. uptake transporters expressed in various organs such as liver, Whereas OATP1B1 and OATP1B3 are predominantly 1,2 kidney, placenta and in the blood-brain barrier. Today, eleven expressed in human liver, other family members such as OATP2B1 human OATP family members have been identified and grouped and OATP4A1 seem to be ubiquitously expressed (for detailed into six different subfamilies (OATP1–OATP6). They mediate description of the tissue expression of human OATP family mem- the sodium-independent uptake of a broad range of amphipatic bers see the review in reference 2). In addition to the expression organic anions from the extracellular environment into cells. in normal, non-malignant tissues, OATPs are present in several Substrates for human OATPs include endogenous substances tumorous tissues and cancer cell lines. OATP1B1 and OATP1B3 such as hormones or hormone metabolites, bile salts and xeno- have been detected in hepatocellular carcinomas, whereas no biotics including several widely prescribed drugs such as HMG- expression of these transporters was found in liver metastases of CoA-reductase inhibitors (statins), antihypertensive drugs and colorectal and pancreatic adenocarcinoma. OATP1B3 seems 1,3,4 11 antibiotics. Furthermore, recent studies have shown that to be overexpressed in colorectal adenocarcinomas. In human several anticancer agents are substrates for some human OATP glioma specimens mRNA expression of OATP1A2, OATP1C1, family members. Atrasentan and methotrexate are transported OATP2B1 and OATP4A1 has been detected. *Correspondence to: Jörg König; Email: joerg.koenig@pharmakologie.med.uni-erlangen.de Submitted: 09/29/10; Revised: 12/15/10; Accepted: 12/19/10 DOI: 10.4161/cbt.11.6.14533 584 Cancer Biology & Therapy Volume 11 Issue 6 Resea RCh pape R Resea RCh pape R Controversial data have been published for the expression demonstrating that only OATP2B1, OATP3A1 and OATP5A1 of OATPs in normal human mammary epithelial cells and mRNA were expressed in adequate amounts when compared to breast cancer. In mammary epithelia cells mRNA expression of the expression of the housekeeping gene b-actin. Primer pairs OATP1A2, OATP2B1, OATP3A1 and OATP4A1 was detected and amplified fragment length are shown in Table 1 . Whereas and the expression of OATP1A2, OATP3A1 and OATP4A1 was these three OATPs were expressed with percentage values (related 14,15 also found in T-47D and MCF-7 breast cancer cell lines. to b-actin) of more than 0.1%, all other OATP family members Interestingly, Pizzagalli and coworkers confirmed the mRNA have expression values below 0.1% b-actin expression suggesting expression of OATP3A1 and OATP4A1, but could not detect a that only OATP2B1, OATP3A1 and OATP5A1 are of significant significant OATP1A2 mRNA expression in normal breast tis- interest in non-malignant and malignant breast tissue. 16 17 sue. In human breast cancer, one study found an overexpres- Next, we investigated the mRNA expression of these three sion of OATP1A2 mRNA compared to corresponding normal uptake transporters in ten normal mammary tissue samples and mammary tissue, whereas no OATP1A2 mR NA expression could in matched samples from the corresponding breast cancer from be detected in a study analyzing the mRNA expression of all the same patient. Clinical characteristics of patients are summa- OATP family members in 13 breast cancer samples. This study rized in Table 2. OATP2B1, OATP3A1 and OATP5A1 mRNA demonstrated the mRNA expression of OATP2A1, OATP2B1, were highly expressed in normal mammary tissue and in matched OATP3A1, OATP4A1, OATP4C1 and OATP5A1, whereas breast cancer with average expression values of 1.4% of b-actin expression of OATP1A2, OATP1B1, OATP1B3, OATP1C1 and mRNA expression for OATP2B1 [95% CI: 0.8–2.0], 3.9% of OATP6A1 was below the detection limit. A recent study by b-actin mRNA expression for OATP3A1 [95% CI: 2.0–5.7%] Maeda et al. analyzed the expression of OATP family mem- and 12.6% of b-actin mRNA expression for OATP5A1 [95% bers in two subclones of MCF-7 breast cancer cells with dif- CI: 5.3–20.0%]. No significant differences between normal ferent estrone-3-sulfate (E3S) uptake activities, demonstrating mammary tissue and tumor tissue were observed (Fig. 1). that among several candidate transporters OATP1B3 might be Localization of OATP2B1, OATP3A1 and OATP5A1 in responsible for the uptake of E3S into MCF-7 cells. Taking these normal human mammary tissue and breast cancer tissue sam- data together it is evident that the results of the mRNA expres- ples. The localization of OATP2B1, OATP3A1 and OATP5A1 sion data of OATP family members in breast cancer tissue or was analyzed using immunohistochemical (Fig. 2) and immu- breast cancer cell line© s are co2 ntro0 vers1 ial a1 nd h aL ve ta o be sn tudd ied ie n s noflu oB rescei nco e ans alyc ses (ie Fig.n 3). Sc pe ecifici. ty of the respective more detail. Moreover, only the protein expression and localiza- antisera used for these analyses has been verified in immunoblot tion of OATP2B1 has been analyzed so far with a lack of protein experiments using homogenates of stably transfected cell lines data for other OATP family members. expressing OATP family members (data not shown). Do not distribute. Based on these expression data and due to the importance of OATP2B1, OATP3A1 and OATP5A1 were detected by OATPs for the cellular uptake of drugs including chemothera- immunohistochemistry in lobules and ducts and in capillary peutic agents it became evident that the detailed knowledge on endothelium in healthy mammary tissue samples and were found the expression of OATP family members in breast cancer tissue to be localized in the cytoplasm and in the plasma membrane of and even more on the tissue specific localization of the expressed malignant cells (Fig. 2). In accordance with the data from immu- proteins can be important for cancer treatment. Furthermore, nohistochemical analyses, immunofluorescence analyses demon - estrogens and estrogen metabolites are substrates of some OATPs strated (Fig. 3) that OATP2B1, OATP3A1 and OATP5A1 are and since these hormones are important for breast cancer pro- localized in the plasma membrane of epithelial cells of lactiferous gression, presence of OATP uptake transporters in plasma mem- ducts. In corresponding tumorous cells the three proteins were branes may be a prerequisite for the intracellular action of these localized in the plasma membrane and to a major extend in the signalling molecules. Therefore, the aim of this study was to cytoplasm. analyze expression of OATP family members in paired samples of normal mammary tissue and corresponding breast cancer Discussion tissue. Furthermore, the cell specific localization of OATP2B1, OATP3A1 and OATP5A1, all highly expressed in mammary epi- Transporter-mediated uptake from the extracellular space into cells thelial cells and breast cancer was investigated by immunohisto- has been recognized as a fundamental step for endogenous pro- chemistry and immunofluorescence analysis. cesses and for drug disposition. Therefore, the knowledge on the tissue-specic e fi xpression of uptake transporters is important for the Results understanding of physiological and pathophysiological processes as well as for drug therapy. In this manuscript we investigated the Expression of mRNA of OATP family members in normal mRNA expression of all human OATP family members and ana- human mammary tissue and breast cancer tissue samples. First, lyzed the localization of the highly expressed OATPs OATP2B1, the mRNA expression of the human OATP family members OATP3A1 and OATP5A1 both in normal mammary tissue and except of the prostaglandin transporter OATP2A1 (OATP1A2, corresponding breast cancer tissue. The mRNA expression analy- OATP1B1, OATP1B3, OATP1C1, OATP2B1, OATP3A1, ses demonstrated that only OATP2B1, OATP3A1 and OATP5A1 OATP4A1, OATP4C1, OATP5A1 and OATP6A1) in five paired are expressed in significant amounts in normal mammary tissue samples of normal breast tissue and breast cancer was investigated samples and that no differences in the amount of mRNA was www.landesbioscience.com Cancer Biology & Therapy 585 Table 1. s equence of primers used in quantitative real-time pCR Forward primer Reverse primer Fragment length Oa Tp1a 2 5'-TGG CTG CCa Ta C CTG Ga T a Ta T-3' 5'-aa T Ta a GT T GTa Ca G Ca T GT T CTC-3' 492 bp Oa Tp1B1 5'-TGC a CT TGG a GG Ca C CTC a C-3' 5'-CT T Ca T CCa TGa Ca C T TC Ca T T T-3' 359 bp Oa Tp1B3 5'-TCa Ta a a CT CT T TGT TCT CTG Ca a -3' 5'-GT T GGC a GC a GC a T T GTC T TG-3' 482 bp Oa Tp1C1 5'-CCT GGa Ta C a Ta T Ta CT T CTG a G-3' 5'-Ca T GT T TCT aaa GT T Ga G T T T CCT-3' 436 bp Oa Tp2B1 5'-TGC TCa TCC Ta a Ga G Ga G TGa a -3' 5'-CCC aa G a Ca GCT Ca C a CT CG-3' 391 bp Oa Tp3a 1 5'-GT T GGG CT T Ca T CCC TCC a C-3' 5'-T Ta GTC a CT a Ta a aa CGG a CT CC-3' 391 bp Oa Tp4a 1 5'-Ga G a CT GTa GCT GTa TCC CTC-3' 5'-GCG GTG GTC a Ga CGC TGC T-3' 531 bp Oa Tp4C1 5'-CTC CTa Ta a CTG TGT CTa TCC T-3' 5'-CCC a T T TCa CCC T TC T T T Ta C T-3' 394 bp Oa Tp5a 1 5'-Ca a Ca G Ga a TGT GGT GTG Ca G-3' 5'-a GC T TC a GG a GG GCG GCT C-3' 402 bp Oa Tp6a 1 5'-Ga T GCa a a G TGC Ta T aa G T Ta -3' 5'-TCC a GT Ta C aa G TCa GT T TCT T-3' 451 bp Table 2. Tumor characteristics of tissue samples Primary Lymph node Metastasis Estrogen Progesterone Patient no. HER-2 status Histological type a a a tumor stage status status receptor status receptor status 1 pT1b N0 M0 - - - n.a. 2 pT1c N0 M0 - - - n.a 3 pT2 N0 M0 - - - ductal 4 pT4b N1 M1 + + - lobular 5 pT2 N1 M0 + + - lobular 6 pT1c N0 M0 + + - ductal ©2011 Landes Bioscience. 7 pT1c N0 M0 + + - ductal 8 pT2 N0 M0 + + - lobular 9 pT1c N1 M0 + - + ductal Do not distribute. 10 pT2 N0 M0 + + - ductal a th a ccording TNM classification, 6 edition, 2003. n.a., not available. observed between normal mammary tissue and corresponding reported recently that they have obtained two subclones of MCF-7 breast cancer tissue. The expression data are largely in line with cells with different estrone-3-sulfate uptake activities and differ- previously published work, in which OATP2B1, OATP3A1 and entially expressed transporter genes supporting the hypothesis of OATP5A1 mRNA was reported to be highly expressed in malig- different cell sublines. nant and non-malignant breast tissue samples. Furthermore, Since the mRNA expression varies between tissue samples mRNA expression of OATP1A2, OATP4A1 and OATP4C1 has and differs to the expression in cancer cells lines we subsequently 17,18 also been detected in some breast cancer tissue samples. This analyzed the localization of the OATP proteins in mammary could be confirmed in our study, but the expression of these OATPs tissue and corresponding cancer tissue. Therefore, we used was very low compared to the expression of OATP2B1, OATP3A1 immunohistochemical (Fig. 2) and immunofluorescence analy - and OATP5A1. Interestingly, as shown in Figure 1, the interin- ses (Fig. 3) to study the localization of the highly expressed dividual variability of the mRNA expression is very high (95% OATPs OATP2B1, OATP3A1 and OATP5A1 in normal breast CI for OATP5A1: 5.3–20.0% b-actin expression) suggesting that tissue and corresponding malignant tissue samples. For the first interindividual differences in gene regulation exists. A discrepancy time we could demonstrate that OATP3A1 and OATP5A1 are could be observed comparing the mRNA expression data in non- localized in the plasma membrane of epithelial cells of the lactifer- malignant or malignant breast cancer tissue with the expression ous ducts in non-malignant breast tissue and also in the plasma in human breast cancer derived cancer cells (e.g., MCF-7 cells). membrane and to a major extend cytoplasmatic in tumorous cells. Expression of OATP1B3 has been detected in MCF-7 cells, Furthermore, we could confirm the previously published local - whereas no expression of OATP1B3 mRNA has been observed ization of OATP2B1, in the same tissue compartments. These in tissue samples. Furthermore, Wlcek et al. have not detected results are of interest for cancer treatment since anticancer agents OATP1B3 mRNA in MCF-7 cells or in non-malignant MCF-10A used in the treatment of breast cancer have been identie fi d as sub - breast cells suggesting that maybe different sublines of MCF-7 cells strates for OATP family members. Paclitaxel and docetaxel have are present in different laboratories or that cell culture conditions been identie fi d being a substrate for OATP1B3, and it has been may be responsible for altered mRNA expression. Maeda et al. demonstrated that OATP1B3 determines methotrexate sensitivity 586 Cancer Biology & Therapy Volume 11 Issue 6 23 23 28 OATP1B1, OATP1B3, and OATP2B1. These previous results demonstrated that both highly expressed OATP family members OATP2B1 and OATP3A1 could be important for the hormonal regulation of breast cells and may also be involved in the progression of breast cancer due to estrogens. Since hormone receptors are located intracellularly, the uptake of hormones and hormone metabolites is a prerequisite for their subsequent regu- latory properties. Therefore, the association between genetic variations in transporter genes encoding uptake transporters for estrogen and estrogen metabolites with breast cancer risk has been studied recently by Justenhoven et al. Interestingly, they demon- strated that none of the frequent polymorphisms in genes encod- ing OATP1A2, OATP1B1, OATP1B3 and OATP2B1 showed an association with the risk for breast cancer suggesting that other OATPs are important for the uptake of these hormones in normal breast tissue and breast cancer. So far no substrates have been iden- tie fi d for OATP5A1 demonstrating the need of further research regarding the role of this uptake transporter for the transport of endogenous substances or drugs. Taken together we have analyzed the mRNA expression of all human OATP family members in non-malignant and malig- nant breast tissue samples and have compared the mRNA expres- sion of the highly expressed OATPs OATP2B1, OATP3A1 and OATP5A1 between normal mammary tissue and matched samples from corresponding tumorous breast tissue samples. Furthermore, ©2011 Landes the t B issue-i spo ecic l fis ocac lizati ioe n on f thec se te hree u. ptake transporters has been analyzed in normal mammary tissue and correspond- ing breast cancer tissue samples. We could demonstrate that these three uptake transporters are localized in the plasma membrane Do not distribute. of epithelial cells of the lactiferous ducts and in addition in the plasma membrane and to a greater extend intracellularly in breast cancer tissue cells. Since estrogen and estrogen metabolites are sub- strates of OATP2B1 and OATP3A1 these results are important for further studies on the molecular action of these hormones and may be relevant for cancer treatment using anticancer agents being substrates for these uptake transporters. Whether selective estro- gen receptor modulators (SERMs; e.g., Tamoxifen), which behave Figure 1. OATP2B1, OATP3A1 and OATP5A1 expression in normal hu- as estrogen receptor antagonists, are also substrates of the OATP man mammary and breast cancer tissue. Quantitative real-time pCR family members expressed in breast cancer has to be elucidated in demonstrated that OATP2B1, OATP3A1 and OATP5A1 mRNa are highly the future. expressed in normal mammary tissue and in corresponding breast can- cer with average expression values of 1.4% of b-actin for OATP2B1 [95% CI: 0.8–2.0%] (a ), 3.9% of b-actin for OATP3A1 [95% CI: 2.0–5.7%] (B) and Materials and Methods 12.6% of b-actin for OATP5A1 [95% CI: 5.3–20.0%] (C). expression levels of all other human Oa Tp family members were significantly lower. No Antibodies and reagents. The polyclonal antibodies AVE and differences between normal mammary tissue and breast cancer were SSS were raised in rabbits against the COOH-terminal sequences observed, p > 0.05. of OATP2B1 (AVE QQL LVS GPG KKP EDS RV A A 690 –709) and OATP5A1 (SSS ADP GLE ESP A AL EPP S A A 830–848), in gastrointestinal cancers. Moreover, several estrogens and estro- respectively. The polyclonal antibody RTK was raised in guinea gen metabolites have been identie fi d as substrates for OATP family pig against the COOH-terminal sequence of OATP3A1 (RTK members and since the mammary gland is an estrogen-responsive FIY NLE DHE WCE NME SVL A A 690–710). Antibodies SSS tissue, uptake of these hormones are important for lobular devel- and RTK were a kind gift from Prof. Dr. D. Keppler (German opment, regulation of epithelia cell growth or steroid hormone Cancer Research Center, Heidelberg, Germany). Cy3-conjugated 20,21 metabolizing enzymes. Estrone-3-sulfate has been identie fi d as AffiniPure goat anti-rabbit IgG and Cy3-conjugated AffiniPure 22 23 24 25 substrate for OATP1A2, OATP1B1, OATP1B3, OATP1C1, donkey anti-guinea pig IgG were obtained from Dianova, cat# 26 26 26 OATP2B1, OATP3A1 and OATP4A1, whereas dehydroepi- 111-165-144 and cat# 706-165-148, respectively. HRP-labeled androsterone-3-sulfate (DHEAS) is a substrate for OATP1A2, Polymer was from EnVisionTM, Dako, cat# K6061. All other www.landesbioscience.com Cancer Biology & Therapy 587 ©2011 Landes Bioscience. Do not distribute. Figure 2. Immunohistochemistry of Oa Tp2B1 (a and D), Oa Tp3a 1 (B and e) and Oa Tp5a 1 (C and F). Immunohistochemical experiments were per- formed using an antiserum directed against human Oa Tp2B1 (a Ve), an antiserum directed against human Oa Tp3a 1 (RTK) and an antiserum directed against human Oa Tp5a 1 (s ss ). Membranous and cytoplasmatic staining was regarded as positive. In normal tissue Oa Tp2B1, Oa Tp3a 1 and Oa Tp5a 1 were detected in lobules (arrows) and ducts (arrow-heads). a ll three targets showed neither in quantity nor in intensity differences between normal tissue (a –C) and breast cancer (D–F). Bars correspond to 100 μm. Figure 3 (See opposite page). Localization of Oa Tp2B1, Oa Tp3a 1 and Oa Tp5a 1 in human normal breast tissue and breast cancer. Oa Tp2B1 is localized in the plasma membrane of epithelial cells of the lactiferous duct and in capillary endothelium. Oa Tp3a 1 and Oa Tp5a 1 are localized in the plasma membrane of epithelial cells of the lactiferous duct. In breast cancer Oa Tp2B1, Oa Tp3a 1 and Oa Tp5a 1 are localized in the plasma membrane and to a major extend in the cytoplasm. 588 Cancer Biology & Therapy Volume 11 Issue 6 ©2011 Landes Bioscience. Do not distribute. Figure 3. For figure legend, see page 588. www.landesbioscience.com Cancer Biology & Therapy 589 chemicals and reagents, unless stated otherwise, were obtained with 2% bovine serum albumin and incubated with a polyclonal from Carl Roth GmbH + Co. KG, Karlsruhe Germany and were rabbit antiserum directed against human OATP2B1 (AVE), a of the highest grade available. polyclonal guinea pig anti-human OATP3A1 (RTK) or a poly- Tissue samples. Human normal and tumorous breast tissue clonal rabbit anti-human OATP5A1 antibody (SSS). Whereas was obtained from patients with incident invasive breast cancer the AVE antiserum is able to detect OATP2B1 proteins encoded undergoing routine primary surgery between 2002–2006 at the by the reference sequence and by known splice variants, the RTK Department of Gynaecology and Obstetrics, Erlangen University antiserum detects only the OATP3A1 protein encoded by the ref- Hospital, Erlangen, Germany. Use of the tissues was approved by erence sequence. Cy3-conjugated AffiniPure donkey anti-guinea the Ethics Committee at the Friedrich-Alexander-University of pig and goat anti-rabbit IgG were used as secondary antibodies. Erlangen-Nuremberg. All patients gave a written informed con- Nuclei were counterstained with SYTOX Green dye (Invitrogen sent. Tissues were collected at the Department of Gynecology and GmbH, cat# S7020). Samples were fixed with cover slips and an Obstetrics (Erlangen, Germany) and classie fi d by the Institute of aqueous mounting medium (Thermo Scientific, cat# 9990402). Pathology, Friedrich-Alexander-University Erlangen-Nuremberg, A confocal laser scanning microscope Axiovert 100M (Carl Erlangen, Germany. Paraffin-embedded tissue samples were Zeiss GmbH, Jena, Germany) was used to visualize the fluores - obtained by the Institute of Pathology (Erlangen, Germany). cence. Images were further processed using the Zeiss LSM Image RNA extraction. Total RNA was isolated from frozen tissue Browser version 4.0.0.157. samples using RNeasy Mini Kit (Quiagen GmbH, cat# 74104) Immunohistochemical analysis. The paraffin sections were according to the manufacturer’s instructions. Concentration and deparaffinized with xylene and rehydrated with graded ethanol. purity of RNA samples was determined by UV absorbance. Slides were subjected to a 5 min heating at 120°C in Tris-EDTA- Quantitative real-time PCR. Messenger-RNA was reverse Buffer pH 8.5 in a pressure cooker. Endogenous peroxidase activity transcribed to cDNA using oligo(dT ) primers and Affinity was quenched by a five min incubation with Peroxidase-Blocking TM Script Multiple Temperature Reverse Transcriptase (Stratagene, solution, Dako REAL (Dako, cat# S2023). Afterwards the sec- cat# 200436). The amount of mRNA was measured by tions were incubated with the polyclonal rabbit antiserum directed quantitative real-time PCR using the LightCycler 2 System against human OATP2B1 (AVE), the purie fi d polyclonal guinea (Roche Diagnostics GmbH) and normalized to the amount pig antiserum directed against human OATP3A1 (RTK) or the of the mRNA of th© e hous2 ekee0 ping g1 en1 e b -L actia n. Tn he ed xpere i-s pol ycB lonal rio abbit as nc tiseri um den irectc ed ae gain. st human OATP5A1 ments were performed using the LightCycler FastStart DNA (SSS) for 30 min. Slides were subsequently incubated with the Plus TM Master SYBR Green I reagents (Roche, cat# 03515885001) HR P-labeled Polymer EnVision + Dual Link (Dako, cat# K4061) and the primers pairs described in Table 1. All primer pairs for 30 min. Antibody binding was visualized using 3,3'-diamino- Do not distribute. are specific for detecting the respective OATP family member. benzidine in chromogen solution, Liquid DAB + Substrate (Dako, For OATP2B1, the primer pair detects all described splice vari- cat# K3467). Tissue sections were counterstained with hematoxy- ants whereas for OATP3A1 only the cDNA of the reference lin (Merck) to visualize tissue structures. The specic fi ity of immu - sequence (NM_013272.3) was detected. Amplification of PCR noreactive signals for OATP2B1, OATP3A1 and OATP5A1 was fragments was started by a denaturation step of 10 min at 95°C, verie fi d by negative controls, which were incubated without the followed by 45 cycles of denaturation at 95°C for 10 s, anneal- respective primary antiserum. ing at 64°C and elongation for 30 s at 72°C. At the end of DNA Statistical analysis. The expression of OATP2B1, OATP3A1 amplification, analyses of melting curves and agarose gel elec - and OATP5A1 mRNA in healthy mammary tissue and corre- trophoresis analyses were performed. sponding cancer tissue samples was analyzed using Mann-Whitney Immunoufl orescence analysis. Cryosections of healthy test. A p value < 0.05 was considered statistically signic fi ant. mammary tissue and tumorous tissue (5 μm thickness) were Acknowledgements prepared using the 2800 Frigocut N (Reichert Jung, now Leica Microsystems Nussloch GmbH). Tissues were fixed in ice-cold The authors’ work is supported by a grant from the Deutsche 70% methanol for 10 min. Subsequently, samples were permea- Krebshilfe (107854) as well as by a grant from the Deutsche bilized using TBS/Triton for 10 min. The tissues were blocked Forschungsgemeinschaft (DFG Ko 2120/1–3). 590 Cancer Biology & Therapy Volume 11 Issue 6 11. Lee W, Belkhiri A, Lockhart AC, Merchant N, Glaeser 21. Katzenellenbogen BS, Montano MM, Ediger TR, Sun References H, Harris EI, et al. Overexpression of OATP1B3 J, Ekena K, Lazennec G, et al. Estrogen receptors: 1. König J, Seithel A, Gradhand U, Fromm MF. confers apoptotic resistance in colon cancer. Cancer selective ligands, partners and distinctive pharmacol- Pharmacogenomics of human OATP transport- Res 2008; 68:10315-23; PMID: 19074900; DOI: ogy. Recent Prog Horm Res 2000; 55:163-93; PMID: ers. 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Lab Invest 2003; 83:527-38; PMID: 12695556. www.landesbioscience.com Cancer Biology & Therapy 591

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