Vaidyanathan Jaya Bharathi, Walle Thomas
Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250505, Charleston, SC 29425, USA.
J Pharmacol Exp Ther. 2003 Nov;307(2):745-52. doi: 10.1124/jpet.103.054296. Epub 2003 Sep 11.
(-)-Epicatechin gallate (ECG) is one of the flavonoids in green tea, which has been demonstrated to have cancer-preventive properties in many model systems. However, the extent and mechanisms of accumulation of these flavonoids in cells is unknown. The objectives of this study were to determine the accumulation of ECG by the intestinal epithelial cell Caco-2 and to characterize the transport mechanism involved. The cells were exposed to ECG +/- various transport inhibitors and incubated at 37 degrees C. Absorbed flavonoids were extracted and quantified by high-performance liquid chromatography. The uptake of ECG included a nonsaturable initial rapid process as well as a much slower saturable process. The saturable ECG uptake by the Caco-2 cells was sodium-independent but clearly dependent on a pH gradient. Phloretin and benzoic acid, inhibitors of the monocarboxylate transporter (MCT), significantly reduced ECG uptake. The uptake of ECG in the Caco-2 cells increased 2-fold in the presence of 50 microM 3-[(3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl)-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK-571), suggesting the involvement of multidrug-associated protein (MRP)2 in efflux of ECG. This was confirmed using Madin-Darby canine kidney cells transfected with MRP2. Also P-glycoprotein was responsible for some ECG efflux. MK-571 also caused a dramatic increase in ECG accumulation in Chinese hamster ovary cells, suggesting that ECG was also a substrate for MRP1. Together, these observations demonstrate important roles of membrane transporters, i.e., MCT, MRP2, P-glycoprotein, and MRP1, in the cellular accumulation and potential effects of ECG.
（-）-表没食子儿茶素没食子酸酯（ECG）是绿茶中的类黄酮之一，在许多模型系统中已证明其具有防癌特性。然而，这些类黄酮在细胞中的积累程度和机制尚不清楚。本研究的目的是确定肠上皮细胞Caco-2对ECG的积累情况，并表征其涉及的转运机制。将细胞暴露于ECG ± 各种转运抑制剂中，并在37℃下孵育。吸收的类黄酮通过高效液相色谱法提取和定量。ECG的摄取包括一个非饱和的初始快速过程以及一个慢得多的饱和过程。Caco-2细胞对ECG的饱和摄取不依赖于钠，但明显依赖于pH梯度。单羧酸转运体（MCT）的抑制剂根皮素和苯甲酸显著降低了ECG的摄取。在存在50μM 3-[(3-[2-(7-氯喹啉-2-基)乙烯基]苯基)-(2-二甲基氨基甲酰基乙基硫烷基)甲基硫烷基]丙酸（MK-571）的情况下，Caco-2细胞中ECG的摄取增加了2倍，这表明多药相关蛋白（MRP）2参与了ECG的外排。使用转染了MRP2的Madin-Darby犬肾细胞证实了这一点。此外，P-糖蛋白也参与了部分ECG的外排。MK-571还导致中国仓鼠卵巢细胞中ECG的积累显著增加，这表明ECG也是MRP1的底物。总之，这些观察结果证明了膜转运体，即MCT、MRP2、P-糖蛋白和MRP1，在ECG的细胞积累和潜在作用中的重要作用。
