Nilotinib (AMN107, Tasigna®) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters
Graphical abstract
Introduction
One of the primary impediments to the successful treatment of cancer is the presence of cells that have acquired the multidrug resistance (MDR) genotype [1], [2]. MDR is often a result of the overexpression of some ATP-dependent transporters that are known as ATP-binding cassette (ABC) transporters. ABC transporters are ubiquitous, in which the most diverse and the largest superfamily of transporters are present on plasma membranes. A majority of the 49 ABC transporters are classified on the basis of two highly conserved nucleotide-binding domains (NBDs), which are located on the cytosolic site [3]. These NBDs have conserved Walker A and B consensus sequences of 90–110 amino acids, where ATP binds and is hydrolyzed via an ATPase enzyme [4]. The energy derived from ATP regulates the transport of various organic molecules, such as vitamins, saccharides, proteins, and several hydrophobic drugs, both intracellularly and extracellularly [5]. It is well established that these ABC transporters, particularly the ABC transporter-subfamily B member 1 (ABCB1/P-gp/MDR1), -subfamily G member 2 (ABCG2/BCRP/MXR/ABCP), and -subfamily C member 1 (ABCC1/MRP1), play a crucial role in the efflux of important chemotherapeutic agents from cells, thereby, inducing MDR [1], [2], [3], [4], [5], [6], [7]. ABCB1 and ABCC1 can also transport various hydrophobic drugs, and ABCC1 can transport some anionic drugs or drug conjugates, including antifolates, certain nucleotides, and also vinca alkaloids [1], [7]. The substrate profile of ABCG2 partially overlaps with that of ABCC1 and ABCB1 tansporters, whose substrates include mitoxantrone (MX), methotrexate (MTX), indolocarbazole topoisomerase I inhibitors, anthracyclines, flavopiridols, daunorubicin, and some camptothecin-derived inhibitors [8]. Also, ABC transporters affect drug disposition by altering absorption, distribution, metabolism, and excretion, as well as their toxicity [9], [10], [11].
In the human genome, more than 500 protein kinases have been identified as molecular switches that catalyze the transition between active and inactive states [12]. Most of these receptors and cytoplasmic protein kinases belong to the serine/threonine and tyrosine kinase families, which play an important role in cellular signaling. Of the 90 genes that code for tyrosine kinases, 32 genes encode non-receptor entities, while the remaining 58 genes encode receptor protein tyrosine kinases (PTKs) [12], [13]. The deregulation of PTKs has been implicated in the development and progression of various solid tumors and hematological malignancies [14]. Recently, a number of tyrosine kinase inhibitors (TKIs) have received FDA approval for the treatment of various types of cancer. Mechanistically, ATP-competitive TKIs directly inhibit autophosphorylation of key tyrosine residues located in the protein kinase activation-loop domain, with subsequent inhibition of substrate phosphorylation and signal transduction. Through binding within a deep cleft between the C-terminal and N-terminal lobes of the conserved kinase domains, TKIs inhibit the access of MgATP to its binding site, thereby preventing the transfer of a phosphate group to a substrate tyrosine residue subsequently abrogating the enzyme's catalytic function in mediating intracellular signaling [15]. There are a significant number of studies indicating that the currently approved TKIs act as anticancer drugs by inhibiting cancer development, proliferation, invasion, metastasis, angiogenesis, and the induction of apoptosis [16], [17].
Currently, no drug has been clinically approved for the reversal of MDR due to detrimental pharmacokinetic interactions, including toxicity issues. Recently, it has been reported that certain clinically used TKIs interact with ABCB1 and ABCG2 transporters [18], [19], [20], [21], [22], [23], [24], [25]. Since hydrophobic TKIs target the intracellular PTK domain, it was hypothesized that some transmembrane transporters, such as ABC transporters, might also modulate the pharmacological activity of specific TKIs [18]. Interestingly, in vitro studies have shown that submicromolar concentrations of several 4-anilinoquinazoline-derived TKIs, such as canertinib, EKI-485, gefitinib, tyrphostin AG1478, erlotinib, lapatinib, and a phenylamino-pyrimidine derivative imatinib (Fig. 1) can selectively modulate MDR protein–ATPase activity, inhibit MDR-dependent active drug efflux, and significantly affect the drug resistance patterns in cells with MDR mediated by ABCB1 and ABCG2 transporters [18], [19], [20], [21], [22], [23], [24], [25]. In vivo studies have suggested that the co-administration of gefitinib enhances oral bioavailability and hence antitumor activity of irinotecan [26]. Gefitinib also decreases the clearance and increases the oral absorption of topotecan by modulating ABCB1 and ABCG2 function in mice [27].
Previously, we have shown that the response to paclitaxel is augmented by lapatinib in ABCB1-overexpressing KBv200 cell xenografts in nude mice [24]. Imatinib has been reported to inhibit ABCG2, enhancing the efficacy of photodynamic therapy by increasing the intracellular accumulation of photosensitizers [28]. In addition, imatinib can significantly reverse topotecan and SN-38 resistance in vitro[29]. The inhibition of ABCB1 and ABCG2 improves the pharmacokinetic delivery of imatinib in brain tumors of mice [30].
Nilotinib (AMN107, Tasigna®) is a phenyl-amino-pyrimidine derivative (Fig. 1), which was designed based on the crystal structure of imatinib–Abl complex. Nilotinib is a potent, relatively selective inhibitor of the tyrosine kinase activities of BCR-Abl, platelet-derived growth factors (PDGFR) and mast/stem-cell growth factor (c-KIT) [31]. A previous study suggested that nilotinib has a high affinity for ABCG2 and that nilotinib is likely to be a substrate of ABCG2 [32]. In addition, a recent study has suggested that nilotinib may also be a substrate of ABCB1 [33]. However, there are no published data indicating if nilotinib can be used to modulate and reverse ABCG2- and ABCB1-mediated MDR. Therefore, in this study, we have sought to determine if nilotinib could modulate ABCB1- and ABCG2-mediated resistance.
Section snippets
Materials
[3H]-MTX (23 Ci/mmol), [3H]-paclitaxel (37.9 Ci/mmol), and [3H]-MX (4 Ci/mmol) were purchased from Moravek Biochemicals, Inc. (Brea, CA). Monoclonal antibodies BXP-34 (against ABCG2) and C-219 (against ABCB1) were acquired from Signet Laboratories, Inc. (Dedham, MA). Anti-actin monoclonal antibody (sc-8432) was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Nilotinib was obtained as a gift from Novartis pharmaceuticals (Basel, Switzerland). Fumitremorgin C (FTC) was synthesized by
Nilotinib significantly enhances the sensitivity of ABCB1- and ABCG2-overexpressing cells to antineoplastic drugs and does not effect the expression of both ABCB1 and ABCG2
Prior to determining the effects of nilotinib to reverse MDR, we examined its effect on the cell viability. The IC50 values of nilotinib alone in ABCG2- and ABCB1-overexpressing cells were ∼10 and ∼30 μM, respectively (data not shown). However, to avoid toxicity in subsequent experiments, the highest concentration of nilotinib used in the reversal experiments was 2.5 μM, a concentration that caused <10% inhibition of growth in all the cell lines used in this study.
Subsequently, we determined
Discussion
Previously, it has been shown that intracellular accumulation of imatinib in the tumor cells was significantly reduced on chronic exposure due to the induction of ABCB1 and ABCG2 transporters, thus leading to pharmacokinetic resistance by decreasing the oral bioavailability of imatinib [40], [41]. In fact, it was seen that the distribution of imatinib in the brain is limited by ABCB1 mediated efflux [30], and that ABCB1 function is responsible for CML resistance on chronic treatment with
Acknowledgements
We are thankful to Novartis for providing us nilotinib. We thank Drs. Michael M. Gottesman (NCI, NIH, Bethesda, USA) for KB-3-1 cells, Shin-ichi Akiyama (Kagoshima University, Japan) for KB-C2 and KB-CV60 cell lines, Susan E. Bates and Robert W. Robey (NIH, USA) for FTC and ABCG2-overexpressing cell lines; Angela Aliberti, Tong Shen (St. John's University) and Yang-min Chen (Mimi) (Montgomery High School, New Jersey) for editorial assistance.
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Grant support: This work was supported by funds from St. John's University Tenure Track Faculty Start-Up Funding No. C-0531 (Z.S. Chen) and St. John's University Seed Grant No. 579-1110 (Z.S. Chen).