Phytomedicine 19 (2012) 977–987 Contents lists available at SciVerse ScienceDirect Phytomedicine j ourna l ho mepage: www.elsevier.de/phymed Carotenoids reverse multidrug resistance in cancer cells by interfering with ABC-transporters Safaa Yehia Eid a , Mahmoud Zaki El-Readi a,b , Michael Wink a,∗ a Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, ImNeuenheimer Feld 364, 69120 Heidelberg, Germany b Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, 71524 Assiut, Egypt a r t i c l e i n f o Keywords: -Carotene Fucoxanthin Canthaxanthin Reversal agents P-glycoprotein Synergy a b s t r a c t Proteins of the ATP-binding cassette superfamily, mainly P-glycoprotein (P-gp; MDR1), play an important role in the development of multidrug resistance (MDR) in cancer cells and thus in the potential failure of chemotherapy. A selection of carotenoids (-carotene, crocin, retinoic acid, canthaxanthin, and fucox- anthin) was investigated whether they are substrates of P-gp, and if they can reverse MDR in resistant Caco-2 and CEM/ADR5000 cells as compared to the sensitive parent cell line CCRF-CEM. The activity of ABC transporter was determined in resistant and sensitive cells by spectrofluorometry and flow cytome- try using the substrates doxorubicin, rhodamine 123, and calcein as fluorescent probes. The carotenoids increased accumulation of these P-gp substrates in a dose-dependent manner indicating that they them- selves also function as substrates. Fucoxanthin and canthaxanthin (50–100 M) produced a 3–5-fold higher retention of the fluorescent probes than the known competitive inhibitor verapamil. Carotenoids showed a low cytotoxicity in cells with MDR with IC 50 values between 100 and 200 M. The combination of carotenoids with eight structurally different cytotoxic agents synergistically enhanced their cytotoxi- city in Caco-2 cells, probably by inhibiting the function of the ABC transporters. For example, fucoxanthin synergistically enhanced the cytotoxicity of 5-FU 53.37-fold, of vinblastine 51.01-fold, and of etoposide 12.47-fold. RT-PCR was applied to evaluate the mRNA levels of P-gp in Caco-2 cells after treatment with carotenoids. Fucoxanthin and canthaxanthin significantly decreased P-gp levels to 12% and 24%, respec- tively as compared to untreated control levels (p < 0.001). This study implies that carotenoids may be utilised as chemosensitisers, especially as adjuvants in chemotherapy. © 2012 Elsevier GmbH. All rights reserved. Introduction Cancer cells often develop cross-resistance to structurally and functionally unrelated cytostatic drugs, a finding which has been termed multidrug resistance (MDR). MDR is a major cause of chemotherapy failure (Baguley 2010). The activity of ATP-dependent multidrug transporters, which belong to the super- family of ATP-binding cassette (ABC) proteins, is important in this context. P-glycoprotein (P-gp = MDR1) is the most widely known ABC-transporter associated with clinical MDR. The enhanced activ- ity of ABC transporters prevent that a toxic concentration of chemotherapeutic drugs is built up in cancer cells. A combina- tion of cytotoxic drugs with non-toxic ABC transporter inhibitors ∗ Corresponding author at: Institut für Pharmazie und Molekulare Biotechnologie, Universität Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany. Tel.: +49 6221 54 4880; fax: +49 6221 54 4884. E-mail addresses: S.Eid@stud.uni-heidelberg.de (S.Y. Eid), wink@uni-hd.de (M. Wink). (chemosensitisers) represents a new approach to overcome drug resistance (Gottesman et al. 2002). Carotenoids are natural tetraterpenes (C40) which are produced by microorganisms, plants, and marine invertebrates. Structurally, carotenoids are derived from the acyclic precursor (,-carotene) by hydrogenation, dehydrogenation, cyclisation, oxidation, or by a combination of these processes. This class of natural products includes carotenes (e.g. -carotene), xanthophylls (e.g. crocin, can- thaxanthin and fucoxanthin), and retinoids (e.g. retinoic acid). More than 700 carotenoids have been described from nature and many carotenes and xanthophylls are typical ingredients of the human diet (Kotake-Nara and Nagao 2011). Recent studies have focused on the biological activities of carotenoids. Carotenoids exhibit anticancer and cancer preven- tive activities by different mechanisms including: antioxidative and direct antiproliferative properties (induction of apoptosis) and by the inhibition of the expression of oncogenes and angiogen- esis (Garattini et al. 2007). Some carotenoids are substrates for ABC transporters, showing high affinity to P-gp in mice lymphoma cells (Shen et al. 2003; Gyemant et al. 2006). However, the same carotenoids did not produce comparable results in several human 0944-7113/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2012.05.010