Kinetic analysis of calcein and calcein -: Acetoxymethylester efflux mediated by the multidrug resistance protein and P-glycoprotein
Kinetic analysis of calcein and calcein -: Acetoxymethylester efflux mediated by the multidrug resistance protein and P-glycoprotein
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DOI：
10.1021/bi9718043
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发表时间：
1998-02-24
期刊：
影响因子：
2.9
通讯作者：
Garnier-Suillerot, A
中科院分区：
文献类型：
--
作者：
Essodaïgui, M;Broxterman, HJ;Garnier-Suillerot, A
多药耐药蛋白（MRP）和P-糖蛋白（Pgp）均属于ATP结合盒质膜药物转运蛋白超家族成员，可能与肿瘤细胞的多药耐药有关。虽然MRP已被确定为有机阴离子转运蛋白和Pgp作为某些带正电荷的化合物的转运蛋白，但在耐药谱中存在相当大的重叠，表明这两种蛋白质转运重要的抗癌药物，如阿霉素、依托泊苷和长春新碱。为了获得更多的见解，在处理药物的两种蛋白质，我们进行了出轨的动力学分析的钙黄绿素乙酰氧基甲酯（GAL-AM），一个共同的中性底物的两种蛋白质的流出，并比较它与钙黄绿素（GAL），这是唯一流出的MRP流出的动力学。GAL是非荧光GAL-AM的水解产物，带负电荷且具有高荧光性。为此，将Pgp+ K562/ADR和MRP+ GLC 4/ADR肿瘤细胞与GAL-AM在富含ATP或耗尽ATP的缓冲液中孵育，并及时观察钙黄绿素的形成和荧光的发展。细胞内的GAL可以区分从流出（细胞外）CAL通过添加到介质中的Co 2+，这完全淬灭细胞外CAL的荧光。结果表明，CAL-AM和GAL通过MRP外排的V-max非常相似（1.0-1.2 × 10（5）分子/细胞/s），但CAL-AM的K-m（0.05 μ M）远低于CAL（268 μ M）。因此，MRP对后者的运输效率远低于CAL-AM。Pgp（0.12 μ M）对CAL-AM转运的Km值与MRP相似。与先前发表的蒽环类药物的数据相比，MRP介导的CAL-AM泵送的动力学数据与中性羟基柔红霉素的动力学数据最相似。这些数据给出了两个相关的，但带不同电荷的化合物的MRP的传输特性的定量帐户。
Multidrug resistance protein (MRP) and P-glycoprotein (Pgp) are both members of the superfamily of ATP binding cassette plasma membrane drug transport proteins, which may be partly responsible for multidrug resistance of tumor cells. Although MRP has been identified as an organic anion transporter and Pgp as a transporter of certain positively charged compounds, there is considerable overlap in resistance spectrum, suggesting that both proteins transport important anticancer agents such as doxorubicin, etoposide, and vincristine. To obtain more insight in the handling of drugs by both proteins, we performed a derailed kinetic analysis of the efflux of calcein-acetoxymethyl eater (GAL-AM), a common neutral substrate for both proteins and compared it with the kinetics of efflux of calcein (GAL) which is only effluxed by MRP. GAL, the hydrolysis product of the nonfluorescent GAL-AM, is negatively charged and highly fluorescent. For this purpose Pgp+ K562/ADR and MRP+ GLC4/ADR tumor cells were incubated with GAL-AM in ATP-rich or ATP-depleted buffer, and the calcein formation was followed in time by fluorescence development. The intracellular GAL could be distinguished from effluxed (extracellular) CAL by addition to the medium of Co2+, which completely quenched the extracellular CAL fluorescence. The results showed that the V-max,,, for efflux of CAL-AM and GAL by MRP were very similar (1.0-1.2 x 10(5) molecules/cell/s) but that the K-m,for CAL-AM was much lower (0.05 mu M) than for CAL (268 mu M). The latter therefore is much less efficiently transported by MRP than CAL-AM. The K-m for CAL-AM transport by Pgp (0.12 mu M) was similar to that for MRP. Compared to previously published data for anthracyclines, the kinetic data for MRP-mediated CAL-AM pumping are most similar to those for the neutral hydroxydaunorubicin. These data give a quantitative account of transport properties of MRP for two related but differently charged compounds.