Nozawa Takashi, Suzuki Masato, Takahashi Koichi, Yabuuchi Hikaru, Maeda Tomoji, Tsuji Akira, Tamai Ikumi
Department of Molecular Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamasaki, Noda, Chiba 278-8510, Japan.
J Pharmacol Exp Ther. 2004 Dec;311(3):1032-7. doi: 10.1124/jpet.104.071522. Epub 2004 Aug 24.
Although circulating estrone-3-sulfate is a major precursor of biologically active estrogen, permeation across the plasma membrane is unlikely to occur by diffusion because of the high hydrophilicity of the molecule. The object of this study was to clarify the involvement of specific transporter(s) in the supply of estrone-3-sulfate to human breast cancer-derived T-47D cells, which grow in an estrogen-dependent manner. The proliferation of T-47D cells was increased by the addition of estrone-3-sulfate, or estradiol, to the cultivation medium. The initial uptake rate of estrone-3-sulfate kinetically exhibited a single saturable component, with Km and Vmax values of 7.6 microM and 172 pmol/mg of protein/min, respectively. The replacement of extracellular Na+ with Li+, K+, or N-methylglucamine+ had no effect on the uptake of [3H]estrone-3-sulfate. The uptake was strongly inhibited by sulfate conjugates of steroid hormones, but not by estradiol-17beta-glucuronide. Taurocholate and sulfobromophthalein inhibited the uptake, whereas other tested anionic and cationic compounds did not. The expression of organic anion transporting polypeptides, OATP-D and OATP-E, which are candidate transporters of estrone-3-sulfate, was detected by reverse transcription-polymerase chain reaction analysis, although their actual involvement in the uptake of estrogen remains to be clarified. In conclusion, the uptake of estrone-3-sulfate by T-47D cells was mediated by a carrier-mediated transport mechanism, suggesting that the estrogen precursor is actively imported by estrogen-dependent breast cancer cells.
尽管循环中的硫酸雌酮是生物活性雌激素的主要前体，但由于该分子具有高亲水性，通过扩散穿过质膜的可能性不大。本研究的目的是阐明特定转运蛋白在硫酸雌酮向人乳腺癌来源的T-47D细胞供应中的作用，该细胞以雌激素依赖的方式生长。向培养基中添加硫酸雌酮或雌二醇可增加T-47D细胞的增殖。硫酸雌酮的初始摄取速率在动力学上表现为单一的可饱和成分，Km和Vmax值分别为7.6微摩尔和172皮摩尔/毫克蛋白质/分钟。用Li+、K+或N-甲基葡糖胺+替代细胞外Na+对[3H]硫酸雌酮的摄取没有影响。摄取受到甾体激素硫酸酯结合物的强烈抑制，但不受17β-雌二醇葡糖苷酸的抑制。牛磺胆酸盐和磺溴酞抑制摄取，而其他测试的阴离子和阳离子化合物则没有。通过逆转录-聚合酶链反应分析检测到有机阴离子转运多肽OATP-D和OATP-E的表达，它们是硫酸雌酮的候选转运蛋白，尽管它们在雌激素摄取中的实际作用仍有待阐明。总之，T-47D细胞对硫酸雌酮的摄取是由载体介导的转运机制介导的，这表明雌激素前体被雌激素依赖的乳腺癌细胞主动摄取。
