Received 3 December 2012
Received in revised form 2 June 2013
Accepted 26 July 2013
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), terpenoicls (menthol. aromadendrene,13-sitosterol-0-glucoside, and 13-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 (Rhol 23) 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 [beta]-carotene (40 [micro]M) and sanguinarine (1 [micro]M) significantly inhibited Rho 123 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 [micro]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.
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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 ABCB 1 gene. It plays a crucial role in the development of multidrug resistance (MDR) by effluxing therapeutical agents out of cancer cells (Gottesman et al. 2002). An overexpression of P-gp has been frequently observed in drug resistant 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 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. cloxorubicin), Vinca alkaloids (e.g. vinblastine), epipodophyllotoxins (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 chemotherapeutical agents might be a strategy to counteract MDR in cancer cells (Wink et al. 2012).
There have been several attempts to overcome...