Phytomedicine 21 (2013) 47–61 Contents lists available at ScienceDirect Phytomedicine j ourna l h o mepage: www.elsevier.de/phymed Influence of combinations of digitonin with selected phenolics, terpenoids, and alkaloids on the expression and activity of P-glycoprotein in leukaemia and colon cancer cells Safaa Yehia Eid a,∗ , Mahmoud Zaki El-Readi a,b , Essam Eldin Mohamed Nour Eldin c , Sameer Hassan Fatani c , Michael Wink a,∗ a Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany b Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, 71524 Assiut, Egypt c Department of Biochemistry, Faculty of Medicine, 6707 Umm AI-Qura University, Saudi Arabia a r t i c l e i n f o Article history: Received 3 December 2012 Received in revised form 2 June 2013 Accepted 26 July 2013 Keywords: Digitonin -Carotene Sanguinarine Multidrug resistance Colon cells Leukaemia a b s t r a c t P-glycoprotein (P-gp or MDR1) is an ATP-binding cassette (ABC) transporter. It is involved in the efflux of several anticancer drugs, which leads to chemotherapy failure and multidrug resistance (MDR) in cancer cells. Representative secondary metabolites (SM) including phenolics (EGCG and thymol), terpenoids (menthol, aromadendrene, -sitosterol-O-glucoside, and -carotene), and alkaloids (glaucine, harmine, and sanguinarine) were evaluated as potential P-gp inhibitors (transporter activity and expression level) in P-gp expressing Caco-2 and CEM/ADR5000 cancer cell lines. Selected SM increased the accumulation of the rhodamine 123 (Rho123) and calcein-AM (CAM) in a dose dependent manner in Caco-2 cells, indicating that they act as competitive inhibitors of P-gp. Non-toxic concentrations of -carotene (40 M) and sanguinarine (1 M) significantly inhibited Rho123 and CAM efflux in CEM/ADR5000 cells by 222.42% and 259.25% and by 244.02% and 290.16%, respectively relative to verapamil (100%). Combination of the saponin digitonin (5 M), which also inhibits P-gp, with SM significantly enhanced the inhibition of P- gp activity. The results were correlated with the data obtained from a quantitative analysis of MDR1 expression. Both compounds significantly decreased mRNA levels of the MDR1 gene to 48% (p < 0.01) and 46% (p < 0.01) in Caco-2, and to 61% (p < 0.05) and 1% (p < 0.001) in CEM/ADR5000 cells, respectively as compared to the untreated control (100%). Combinations of digitonin with SM resulted in a significant down-regulation of MDR1. Our findings provide evidence that the selected SM interfere directly and/or indirectly with P-gp function. Combinations of different P-gp substrates, such as digitonin alone and together with the set of SM, can mediate MDR reversal in cancer cells. © 2013 Elsevier GmbH. All rights reserved. Introduction ATP-binding cassette (ABC) transporters are transmembrane proteins, which can transport a wide variety of compounds across cell membranes utilizing the energy from ATP hydrolysis. P- glycoprotein (P-gp or MDR1) is a member of the ABC transporter subfamily B and encoded by the ABCB1 gene. It plays a crucial role in the development of multidrug resistance (MDR) by effluxing the- rapeutical agents out of cancer cells (Gottesman et al. 2002). An overexpression of P-gp has been frequently observed in drug resis- tant tumour cells and has been proposed as a cause for the failure of a broad range of anti-cancer drugs (Dean et al. 2001). Most of ∗ Corresponding authors. Tel.: +49 6221 54 4880; fax: +49 6221 54 4884. E-mail addresses: S.Eid@stud.uni-heidelberg.de, safaamenesi@yahoo.com (S.Y. Eid), wink@uni-hd.de (M. Wink). the commonly used chemotherapeutic agents are P-gp substrates which carry a positive charge (or are neutral) at physiological pH, are cyclic and hydrophobic molecules, such as anthracyclines (e.g. doxorubicin), Vinca alkaloids (e.g. vinblastine), epipodophyllotox- ins (e.g. etoposide), and taxanes (e.g. paclitaxel) (Sauna et al. 2007). Substances that completely, or partly, inhibit P-gp function and/or expression may prevent the undesirable efflux of anticancer agents from MDR cancer cells. Combining P-gp inhibitors with chemother- apeutical agents might be a strategy to counteract MDR in cancer cells (Wink et al. 2012). There have been several attempts to overcome MDR by develop- ing effective P-gp inhibitors. Calcium channel blockers (verapamil), calmodulin inhibitors (phenothiazines), nonpolar cyclic oligopep- tides with immunosuppressant activity (cyclosporin A and the cyclosporin D analogue Valspodar PSC 833) have been identified as P-gp inhibitors (Tan et al. 2000). These inhibitors can success- fully reverse MDR in vitro, but their efficacy in animal studies and 0944-7113/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.phymed.2013.07.019