Nilotinib (TasignaRTM) Modulates ABCB1/PGP- and ABCG2/BCRP-mediated multidrug resistance in cancer chemotherapy.

摘要

It is well established that overexpression of ABC transporters are involved in the extrusion of a variety of therapeutic drugs, leading to multidrug resistance (MDR), which can eventually produce cancer chemotherapy failure. Previously, it was shown that specific tyrosine kinase inhibitors (TKIs) could modulate several human ATP-binding cassette (ABC) proteins. Nilotinib is a second generation BCR-ABL TIC, which was developed to surmount resistance or intolerance to imatinib in patients with Philadelphia chromosome positive chronic myelogenous leukemia (Ph+-CML). Here, we report for the first time that nilotinib not only potentiates the cytotoxicity of widely used therapeutic substrates of ABCB1, such as colchicine, vincristine, and paclitaxel, but also that of ABCG2 substrates including as mitoxantrone (MX) and doxorubicin. Nilotinib significantly enhances the intracellular accumulation of MX in HEK293 cells transfected with ABCG2. Similarly, nilotinib significantly increases the accumulation of paclitaxel in cell lines overexpressing ABCB1. Furthermore, nilotinib produces a concentration-dependent inhibition of the ABCG2-mediated transport of [314]-methotrexate (MTX), as well as [31-1]-E217f3G, a physiological substrate of ABCG2. Uptake studies in membrane vesicles overexpressing ABCG2 have indicated that nilotinib inhibits ABCG2, similar to other established TKIs as well as fumitremorgin C, a known inhibitor of ABCG2. In addition, the kinetic analysis demonstrated that nilotinib is a potent competitive inhibitor of MTX transport by ABCG2 with a Ki value of 0.69+7-0.083 1.1.M. However, Western blot analysis confirmed that nilotinib does not significantly alter the expression of ABCB1 and ABCG2 transporters. In fact, a mechanistic study revealed that nilotinib stimulates ATPase activity of both ABCB1 and ABCG2 at sub-micromolar concentrations. Furthermore, nilotinib directly and potently interacts with ABCB1 and ABCG2 at its drug-binding site as shown by photo affinity labelling assay, where nilotinib competitively displaces [125I]-IAAP from its known substrate binding sites of these transporters. This direct interaction of nilotinib was further confirmed by docking nilotinib within the large cavity of the transmembrane region of ABCB1 and predicting the binding conformation of nilotinib on the homologues human ABCB1 structure designed on mouse ABCB1 crystal structure. Nilotinib was found to bind at the same site as that of IAAP with a high docking -11.55 glides score. Importantly, nilotinib at 75mg/kg strongly enhanced the effect of paclitaxel (18 mg/kg) on the inhibition of growth of the ABCB1-overexpressing epidermoid carcinoma KB-C2 cell xenografts in athymic (nu/nu) BALB/c nude mice. Similarly, nilotinib (75 mg/kg) in combination with doxorubicin (3 mg/kg) significantly sensitized ABCG2-overexpressing non-small cell lung cancer H460/MX-20 cell xenograft in athymic (nu/nu) BALB/c nude mice model. Nilotinib synergistically and significantly decreased the volume of both ABCB1- and ABCG2-overexpressing MDR tumors with no visible toxicity or weight loss. Our results indicate that nilotinib could overcome ABCB1- and ABCG2-mediated MDR by blocking the efflux function of these transporters. These findings may be useful in clinical practice for cancer combination therapy with nilotinib and they may be vital to the design of present and future clinical trials with nilotinib, elucidating potential pharmacokinetic interactions.