Essodaigui M, Broxterman H J, Garnier-Suillerot A
Laboratoire de Physicochimie Biomoleculaire et Cellulaire (URA CNRS 2056), Universite Paris Nord, Bobigny 93017, France.
Biochemistry. 1998 Feb 24;37(8):2243-50. doi: 10.1021/bi9718043.
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 detailed kinetic analysis of the efflux of calcein-acetoxymethyl ester (CAL-AM), a common neutral substrate for both proteins and compared it with the kinetics of efflux of calcein (CAL) which is only effluxed by MRP. CAL, the hydrolysis product of the nonfluorescent CAL-AM, is negatively charged and highly fluorescent. For this purpose Pgp+ K562/ADR and MRP+ GLC4/ADR tumor cells were incubated with CAL-AM in ATP-rich or ATP-depleted buffer, and the calcein formation was followed in time by fluorescence development. The intracellular CAL 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 Vmax for efflux of CAL-AM and CAL by MRP were very similar (1.0-1.2 x 10(5) molecules/cell/s) but that the Km for CAL-AM was much lower (0.05 microM) than for CAL (268 microM). The latter therefore is much less efficiently transported by MRP than CAL-AM. The Km for CAL-AM transport by Pgp (0.12 microM) 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.
多药耐药蛋白（MRP）和P-糖蛋白（Pgp）都是ATP结合盒质膜药物转运蛋白超家族的成员，它们可能部分导致肿瘤细胞的多药耐药。尽管MRP已被鉴定为有机阴离子转运体，Pgp为某些带正电荷化合物的转运体，但耐药谱有相当大的重叠，这表明这两种蛋白都能转运重要的抗癌药物，如阿霉素、依托泊苷和长春新碱。为了更深入了解这两种蛋白对药物的处理过程，我们对钙黄绿素-乙酰氧基甲酯（CAL-AM）的外排进行了详细的动力学分析，CAL-AM是这两种蛋白的常见中性底物，并将其与仅由MRP外排的钙黄绿素（CAL）的外排动力学进行了比较。CAL是无荧光的CAL-AM的水解产物，带负电荷且荧光很强。为此，将Pgp+ K562/ADR和MRP+ GLC4/ADR肿瘤细胞在富含ATP或缺乏ATP的缓冲液中与CAL-AM孵育，并通过荧光发展随时间跟踪钙黄绿素的形成。通过向培养基中添加Co2+，可以将细胞内的CAL与外排（细胞外）的CAL区分开来，Co2+能完全淬灭细胞外CAL的荧光。结果表明，MRP对CAL-AM和CAL的外排Vmax非常相似（1.0 - 1.2×10⁵分子/细胞/秒），但CAL-AM的Km（0.05 microM）比CAL（268 microM）低得多。因此，MRP转运CAL的效率远低于CAL-AM。Pgp转运CAL-AM的Km（0.12 microM）与MRP相似。与先前发表的蒽环类药物数据相比，MRP介导的CAL-AM泵浦的动力学数据与中性羟基柔红霉素的数据最相似。这些数据定量说明了MRP对两种相关但电荷不同的化合物的转运特性。
