Abstract
P-glycoprotein (P-gp) is a membrane protein associated with multidrug resistance (MDR) due to its key role in mediating the traffic of chemotherapeutic drugs outside cancer cells, leading to a cellular response that hinders efforts toward successful therapy. With the aim of finding agents that circumvent the MDR phenotype mediated by P-gp, 15 compounds isolated from native and naturalized plants of Argentina were screened. Among these, the non-cytotoxic lignan (±) pinoresinol successfully restored sensitivity to doxorubicin from 7 μM in the P-gp overexpressed human myelogenous leukemia cells, Lucena 1. This resistance-reversing effect was confirmed by competitively increasing the intracellular doxorubicin accumulation and by significantly inhibiting the efflux of doxorubicin and, to a lesser extent, that of rhodamine 123. The activity obtained was similar to that observed with verapamil. No such results were observed in the sensitive parental K562 cell line. To gain deeper insight into the mode of action of pinoresinol, its effect on P-gp function and expression was examined. The docking simulations indicated that the lignan bound to P-gp at the apex of the V-shaped transmembrane cavity, involving transmembrane helices 4, 5, and 6, and partially overlapped the binding region of tariquidar, which was used as a positive control. These results would shed some light on the nature of its interaction with P-gp at molecular level and merit further mechanistic and kinetic studies. In addition, it showed a maximum 29% activation of ATP hydrolysis and antagonized verapamil-stimulated ATPase activity with an IC50 of 20.9 μM. On the other hand, pinoresinol decreased the presence of P-gp in the cell surface. Derivatives of pinoresinol with improved activity were identified by docking studies. The most promising one, the non-cytotoxic 1-acetoxypinoresinol, caused a reversion of doxorubicin resistance from 0.11 μM and thus higher activity than the lead compound. It also caused a significant increase in doxorubicin accumulation. Results were similar to those observed with verapamil. The results obtained positioned these compounds as potential candidates for effective agents to overcome P-gp-mediated MDR, leading to better outcomes for leukemia chemotherapy.
Highlights
The P-glycoprotein (P-gp) transporter (ABCB1/MDR1) encoded by the multidrug resistance (MDR)-1 gene is a protein located in the cell membranes of various tissues involved in the traffic of substrates outside the cells (Sharom, 2011; Silva et al, 2015)
Since there is no common pharmacophore determining that a compound behaves as a P-gp reverser (Yuan et al, 2012) and due to the wide diversity of chemical structures that interact with this transporter (Robert and Jarry, 2003) as well as the lack of information on metabolites from Argentinian flora as P-gp chemosensitizers, we decided to investigate this effect in 15 plant-derived compounds belonging to different chemical families obtained from plants from central Argentina (Carpinella et al, 2002, 2003, 2005; Diaz Napal et al, 2009; Chiari et al, 2010, 2011; Joray et al, 2011, 2013, 2015; del Corral et al, 2012; Diaz Napal and Palacios, 2013)
Compounds 1–15 were studied by an MTT assay, in order to evaluate the potentiation of DOX cytotoxicity in insensitive Lucena 1 cells, which were 35-fold more resistant to this drug [IC50 = 40.78 (16.60–100.18) μM] than their parental cell line K562 [IC50 = 1.16 (0.52–2.58) μM]
Summary
The P-glycoprotein (P-gp) transporter (ABCB1/MDR1) encoded by the MDR-1 gene is a protein located in the cell membranes of various tissues involved in the traffic of substrates outside the cells (Sharom, 2011; Silva et al, 2015). Each half consists of six transmembrane α-helices (TMHs) and one cytoplasmic nucleotide-binding domain (NBD), which fuel the energy from ATP hydrolysis, leading to conformational changes that result in the extrusion of a set of structurally and functionally unrelated chemotherapy drugs against their concentration gradient (Sharom, 2011). P-gp keeps intracellular drug accumulation low, leading to a cellular responsiveness known as classical MDR (Krishna and Mayer, 2000). This phenomenon and the broad spectrum of substrates removed from cells, such as paclitaxel, etoposide, teniposide, vinblastine, vincristine, doxorubicin, daunorubicin, and imatinib among others (Kathawala et al, 2015), makes this pump one of the most significant transporters in pharmacology
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