Role of Transferrin Receptor and the ABC Transporters ABCB6 and ABCB7 for Resistance and Differentiation of Tumor Cells towards Artesunate Gerhard Kelter1., Daniel Steinbach2., Venkata Badireenath Konkimalla3, Tsuyoshi Tahara4, Shigeru Taketani4, Heinz-Herbert Fiebig1, Thomas Efferth3* 1 Oncotest GmbH, Institute of Experimental Oncology, Freiburg, Germany, 2 Department for Pediatrics, University of Ulm, Ulm, Germany, 3 Pharmaceutical Biology (C015), German Cancer Research Center, Heidelberg, Germany, 4 Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan The anti-malarial artesunate also exerts profound anti-cancer activity. The susceptibility of tumor cells to artesunate can be enhanced by ferrous iron. The transferrin receptor (TfR) is involved in iron uptake by internalization of transferrin and is over- expressed in rapidly growing tumors. The ATP-binding cassette (ABC) transporters ABCB6 and ABCB7 are also involved in iron homeostasis. To investigate whether these proteins play a role for sensitivity towards artesunate, Oncotest’s 36 cell line panel was treated with artesunate or artesunate plus iron(II) glycine sulfate (FerrosanolH). The majority of cell lines showed increased inhibition rates, for the combination of artesunate plus iron(II) glycine sulfate compared to artesunate alone. However, in 11 out of the 36 cell lines the combination treatment was not superior. Cell lines with high TfR expression significantly correlated with high degrees of modulation indicating that high TfR expressing tumor cells would be more efficiently inhibited by this combination treatment than low TfR expressing ones. Furthermore, we found a significant relationship between cellular response to artesunate and TfR expression in 55 cell lines of the National Cancer Institute (NCI), USA. A significant correlation was also found for ABCB6, but not for ABCB7 in the NCI panel. Artesunate treatment of human CCRF-CEM leukemia and MCF7 breast cancer cells induced ABCB6 expression but repressed ABCB7 expression. Finally, artesunate inhibited proliferation and differentiation of mouse erythroleukemia (MEL) cells. Down-regulation of ABCB6 by antisense oligonucleotides inhibited differentiation of MEL cells indicating that artesunate and ABCB6 may cooperate. In conclusion, our results indicate that ferrous iron improves the activity of artesunate in some but not all tumor cell lines. Several factors involved in iron homeostasis such as TfR and ABCB6 may contribute to this effect. Citation: Kelter G, Steinbach D, Konkimalla VB, Tahara T, Taketani S, et al (2007) Role of Transferrin Receptor and the ABC Transporters ABCB6 and ABCB7 for Resistance and Differentiation of Tumor Cells towards Artesunate. PLoS ONE 2(8): e798. doi:10.1371/journal.pone.0000798 INTRODUCTION tumors [18]. The expression of TfR is of prognostic significance Artemisinin is a sesquiterpene isolated from Artemisia annua L., for several tumor types [19–21]. Ferrous iron can either be bound which is used in traditional Chinese medicine for the treatment of to transferrin or to other proteins before uptake. fever and chills [1]. Artemisinin reveals profound activity against The ATP-binding cassette (ABC) transporters ABCB6 and Plasmodium falciparum and Plasmodium vivax [2,3]. Artesunate and ABCB7 are involved in iron homeostasis. They are located in the artemether are semi-synthetic derivatives of artemisinin with mitochondria and transport heme and protoporphyrins into these improved pharmacological features [4]. In addition to their anti- organelles [22]. Most ABC transporters are involved in the active malarial activity, artemisinin and its derivatives are also active transport of phospholipids, ions, peptides, steroids, polysaccha- against cancer cells [5–9]. In both cases, the activity of the drugs is rides, amino acids, bile acids, pharmaceutical drugs and other associated with the presence of iron. Iron is present in large excess xenobiotic compounds [23]. bound to hemoglobin in erythrocytes, where the Plasmodia In humans, 49 different ABC transporters have been identified, parasites are located. The active moiety of artemisinin-like drugs which are classified into seven sub-families (A–G) [24]. In healthy is an endoperoxide bridge, whose reductive homolysis is promoted organs several ABC transporters protect against the harmful effects by iron(II)-heme leading to C4-centered alkylating radicals [10]. These radical molecules cause macromolecular damage by alkylating essential malarial proteins inducing cell death of Academic Editor: Mikhail Blagosklonny, Ordway Research Institute, United States parasites [11–13]. On the other hand, iron content is higher in of America tumor cells than in normal cells [14] making them more Received May 18, 2007; Accepted July 30, 2007; Published August 29, 2007 susceptible to artemisinins. We and others have shown that the susceptibility of tumor cells to artemisinins can further be Copyright: ß 2007 Kelter et al. This is an open-access article distributed under enhanced by the addition of transferrin or ferrous iron [15,16]. the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the The role of artemisinins and iron for malaria treatment has original author and source are credited. been intensively investigated during the past years [17], whereas the role of iron for tumor treatment with artemisinins is far less Funding: This work was funded by intramural grants. understood. The iron-binding protein, transferrin, is internalized Competing Interests: The authors have declared that no competing interests into cancer cells after binding to the transferrin receptor (TfR, exist. CD71). This is a transmembrane glycoprotein involved in iron * To whom correspondence should be addressed. E-mail: [email protected] uptake by internalization of transferrin. TfR exerts growth regulatory functions and is over-expressed in rapidly growing . These authors contributed equally to this work. PLoS ONE | www.plosone.org 1 August 2007 | Issue 8 | e798 Iron and Drug Resistance of xenobiotics taken up with food. A high expression can, Propidium iodide (PI) assay therefore, be found in the gastrointestinal tract, liver, and kidney. A modified propidium iodide assay [35] was used to assess the A protective function is also given as components of the blood compound’s activity in Oncotest’s 36 cell line panel. Briefly, cells brain barrier and the blood placenta barrier. were harvested from exponential phase cultures by trypsinization, ABC transporters have been intensively investigated in the past counted and plated in 96 well flat-bottomed micro-titer plates at years. ABCB1 (P-glycoprotein, MDR1), ABCC1-C6 (MRP1-6) and a cell density depending on the cell line (4.000–10.000 cells/well). ABCG2 (BCRP) confer resistance to cytostatic drugs of tumors After a 24 h recovery period to allow the cells to adhere and and contribute to the failure of tumor chemotherapy [25]. The resume exponential growth, 10 ml of culture medium (six control mutated gene product of ABCC7 (CFTR) is involved in the wells/plate) or of culture medium containing the test compounds pathogenesis cystic fibrosis, and some other mutated ABC were added to the cells. The compounds were applied in triplicates transporter genes contribute to a number of hereditary diseases at five concentrations. Following four days of continuous drug and disorders [24]. The pathogenic function of the majority of exposure, medium or medium with test compound, including all ABC transporters still awaits elucidation. To gain insight into dead cells suspended in the culture medium, was aspirated and functional aspects, we have developed a low density microarray for replaced by 200 ml of an aqueous propidium iodide (PI) solution ABC transporters [26,27]. Applying this microarray, we identified (7 mg/ml). To measure the amount of living cells, cells were ABCA2 and ABCA3 as further genes involved in tumor drug permeabilized by freezing the plates, resulting in the death of all resistance [28,29]. cells that had remained attached to the bottom of the well after the In the present investigation, we focused on TfR, ABCB6, and incubation period. After thawing of the plates, fluorescence was ABCB7. The aim of the present study was to address the question, measured using the Cytofluor 4000 micro-plate reader (excitation whether these proteins are involved in the response of tumor cells 530 nm, emission 620 nm), providing a direct relationship to the to artesunate. total viable cell number. MATERIALS AND METHODS Cell line array Drugs Arrays of cell lines from the Oncotest Human Cell Line Collection Artesunate was obtained from Saokim Ltd. (Hanoi, Vietnam) and were assembled using a tissue arrayer (Beecher Instruments, Sun iron(II) glycine sulfate (FerrosanolH) from Sanol (Munich, Germany). Prairie, Wisc., USA) as recently described [36]. Pellet biopsies (0.6 mm in diameter) were taken from embedded cells and arrayed Cell Lines in duplicate in a new recipient paraffin block. Four-micrometer CCRF-CEM were cultured in RPMI medium and MCF-7 and sections of the resulting microarray block were cut and transferred MEL cells in DMEM medium each supplemented with 10% fetal onto glass slides using the paraffin-sectioning aid system calf serum under standard conditions (37uC, 5% CO2). Cells were (Instrumedics, Hackensack, NJ, USA) and further processed for passaged twice weekly. immunohistochemical staining. The arrays were stained with the