Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding.
ABSTRACT ABCG2 [also known as BCRP (breast cancer resistance protein) or MXR] is an ABC (ATP-binding cassette) protein shown to confer multidrug resistance. ABCG2 was initially identified in resistant breast carcinoma cells (MCF-7/AdrVp1000) selected with doxorubicin and verapamil. Later studies demonstrated the presence of a point mutation (Arg482 to Thr) in ABCG2 in MCF-7/AdrVp1000 cells. This mutation was shown to modulate the transport of Rh123 (rhodamine 123). In the present study, we have used a previously characterized photoreactive drug analogue of Rh123, IAARh123 (iodoaryl-azido-Rh123), to examine the effects of the Arg482Thr mutation on Rh123 binding and transport by ABCG2. Our results show that both wild-type (ABCG2R482) and mutant (ABCG2T482) ABCG2 bound directly to IAARh123. Surprisingly, however, wild-type ABCG2R482, which does not transport Rh123, was more intensely photolabelled than mutant ABCG2T482. In addition, inhibition of IAARh123 photolabelling using various drug substrates of ABCG2 revealed some differences between wild-type and mutant ABCG2. For example, a molar excess of mitoxantrone was more effective at inhibiting IAARh123 labelling of wild-type than of mutant ABCG2, while excess cisplatin, taxol and methotrexate showed significant inhibition of IAARh123 binding to both wild-type and mutant ABCG2. Taken together, the results of this study provide the first demonstration of the direct binding of drugs to ABCG2.
Article: Effects of monoglycerides on rhodamine 123 accumulation, estradiol 17 beta-D-glucuronide bidirectional transport and MRP2 protein expression within Caco-2 cells.[show abstract] [hide abstract]
ABSTRACT: Oral drug development had been hindered by the bioavailability issue despite vast market popularity. Lipid excipients had shown to enhance bioavailability of a number of reformulated hydrophobic oral drugs, yet the underlying mechanisms of action by lipids are still unclear. One proposed mechanism is that lipid excipients could facilitate drug uptake by altering the activities of apical membrane intestinal efflux transporters. Thus, this study aimed to investigate the effects of 1-monopalmitin, 1-monoolein and 1-monostearin on the efflux activity and protein expression of multidrug resistance-associated protein 2 (MRP2) in vitro. The 24-hour non-cytotoxic ranges of these monoglycerides were first determined using MTS and LDH assays in Caco-2 cells. Then, both accumulation and bidirectional transport studies were conducted using 10 microM rhodamine 123 (Rh123) and 10 nM estradiol 17 beta-D-glucuronide (E(2)17betaG), respectively, to assess the functional activities of MRP2. 50 microM MK-571, a specific MRP1 and MRP2 inhibitor, was used as the positive control in both studies. Western blotting was followed to determine the effect of these monoglycerides on MRP2 protein expression. Caco-2 cells were viable when treated with 1-monopalmitin, 1-monostearin and 1-monoolein at concentrations equal or less than 1000 microM, 1000 microM and 500 microM, respectively. Cells treated with 1-monoplamitin, 1-monostearin, 1-monoolein and MK571 resulted in significant increases in Rh123 accumulation and decreases in E(2)17BetaG efflux ratio compared to the control (medium treated only). MRP2 protein expressions in 1-monopalmitin and 1-monoolein treated cells were decreased by 19% and 35% compared to the control; however, there was no change of MRP2 protein expression in 1- monostearin treated cells. These findings suggested that 1-monoolein, 1-monostearin and 1-monopalmitin could attenuate the activity of MRP2 and possibly other efflux transporters in Caco-2 cells. The reduction of efflux activity of MRP2 by 1-monoolein treatment could be partially accounted by the non-specific down-regulation of MRP2 protein expression.Journal of pharmacy & pharmaceutical sciences: a publication of the Canadian Society for Pharmaceutical Sciences, Societe canadienne des sciences pharmaceutiques 02/2008; 11(3):45-62. · 1.65 Impact Factor