Verapamil and Its Derivative Trigger Apoptosis through Glutathione Extrusion by Multidrug Resistance Protein MRP1

Abstract

This study demonstrates that verapamil and a newly synthesized verapamil derivative, NMeOHI₂, behave as apoptogens in multidrug resistance protein 1 (MRP1)-expressing cells. When treated with either verapamil or NMeOHI₂, surprisingly, baby hamster kidney-21 (BHK) cells transfected with human MRP1 were killed. Because parental BHK cells were not, as well as cells expressing an inactive (K1333L) MRP1 mutant, this indicated that cell death involved functional MRP1 transporter. Cell death was identified as apoptosis by using annexin V-fluorescein labeling and was no longer observed in the presence of the caspase inhibitor Z-Val-Ala-Asp(OMe)-CH₂F (Z-VAD-FMK). In vitro, both verapamil and its derivative inhibited leukotriene C4 transport by MRP1-enriched membrane vesicles in a competitive manner, with a K_i of 48.6 μm for verapamil and 5.5 μm for NMeOHI_2, and stimulated reduced glutathione (GSH) transport 3-fold and 9-fold, respectively. Treatment of MRP1-expressing cells with either verapamil or the derivative quickly depleted intracellular GSH content with a strong decrease occurring in the first hour of treatment, which preceded cell death beginning at 8–16 h. Furthermore, addition of GSH to the media efficiently prevented cell death. Therefore, verapamil and its derivative trigger apoptosis through stimulation of GSH extrusion mediated by MRP1. This new information on the mechanism of induced apoptosis of MDR cells may represent a novel approach in the selective treatment of MRP1-positive tumors.