Inhibition of the MRP1-mediated transport of the menadione-glutathione conjugate (thiodione) in HeLa cells as studied by SECM
Inhibition of the MRP1-mediated transport of the menadione-glutathione conjugate (thiodione) in HeLa cells as studied by SECM
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DOI：
10.1073/pnas.1201555109
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发表时间：
2012-07-17
影响因子：
11.1
通讯作者：
Bard, Allen J.
中科院分区：
文献类型：
--
作者：
Koley, Dipankar;Bard, Allen J.
用扫描电化学显微镜（SECM）对甲萘醌（2-methyl-1，4-napthaquinone）诱导的HeLa细胞氧化应激进行了真实的实时研究。疏水性分子甲萘醌通过活细胞膜扩散，在那里它对细胞有毒。然而，在细胞中，它与谷胱甘肽结合形成硫代二酮。然后，硫代二酮被识别并通过ATP驱动的MRP 1泵跨细胞膜转运。在细胞外环境中，硫代二酮检测SECM尖端在140，70，和35 μ M的水平后，暴露的细胞甲萘醌浓度为500，250，和125 μ M，分别。借助于有限元建模，确定了噻二酮转运的动力学为1.6 × 10（-7）m/s，比甲萘醌吸收快约10倍。在500 μ M甲萘醌和50 μ M MK 571存在下，MK 571选择性抑制活HeLa细胞内的这些MRP 1泵产生50 μ M的较低噻二酮浓度。在存在单克隆抗体QCRL-4（一种MRP 1泵的选择性阻断剂）的情况下，观察到类似的噻二酮外排减少（下降50%）。减少的硫代二酮通量证实，硫代二酮转运的MRP 1，谷胱甘肽是一个必不可少的底物MRP 1介导的运输。这一发现证明了SECM在MRP 1抑制剂定量研究中的有用性，并表明单克隆抗体可以成为抑制这些MDR泵转运的有用工具，从而有助于克服多药耐药性。
Oxidative stress induced in live HeLa cells by menadione (2-methyl-1,4-napthaquinone) was studied in real time by scanning electrochemical microscopy (SECM). The hydrophobic molecule menadione diffuses through a living cell membrane where it is toxic to the cell. However, in the cell it is conjugated with glutathione to form thiodione. Thiodione is then recognized and transported across the cell membrane via the ATP-driven MRP1 pump. In the extracellular environment, thiodione was detected by the SECM tip at levels of 140, 70, and 35 mu M upon exposure of the cells to menadione concentrations of 500, 250, and 125 mu M, respectively. With the aid of finite element modeling, the kinetics of thiodione transport was determined to be 1.6 x 10(-7) m/s, about 10 times faster than menadione uptake. Selective inhibition of these MRP1 pumps inside live HeLa cells by MK571 produced a lower thiodione concentration of 50 mu M in presence of 500 mu M menadione and 50 mu M MK571. A similar reduced (50% drop) thiodione efflux was observed in the presence of monoclonal antibody QCRL-4, a selective blocking agent of the MRP1 pumps. The reduced thiodione flux confirmed that thiodione was transported by MRP1, and that glutathione is an essential substrate for MRP1-mediated transport. This finding demonstrates the usefulness of SECM in quantitative studies of MRP1 inhibitors and suggests that monoclonal antibodies can be a useful tool in inhibiting the transport of these MDR pumps, and thereby aiding in overcoming multidrug resistance.