Kishimoto Shuichi, Kawazoe Yuji, Ikeno Mako, Saitoh Mizuha, Nakano Yukari, Nishi Yuko, Fukushima Shoji, Takeuchi Yoshikazu
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, Kobe Gakuin University, Nishi-ku, Kobe, 651-2180 Japan.
Cancer Chemother Pharmacol. 2006 Jan;57(1):84-90. doi: 10.1007/s00280-005-0003-x. Epub 2005 Nov 5.
The present study was undertaken to identify what regulates intracellular cisplatin (CDDP) accumulation and what changes in membrane fraction of CDDDP-resistant cell line. The CDDP-resistant rat hepatoma cell line, H4-II-E/CDDP, shows a significant decrease in intracellular platinum accumulation compared with parental H4-II-E cells, although there was no difference in the efflux of CDDP between these two cell lines. In this study, we examined the contribution of functional change in active transport to the CDDP resistance of H4-II-E/CDDP cells. Compared with the resistant cells, platinum accumulation in the parental cells was clearly decreased by low temperature or ATP depletion. In addition, the Na+, K+-ATPase inhibitor ouabain and the K+ channel inhibitor tetraethylammonium decreased platinum accumulation in parental cells but did not change the accumulation in resistant cells. Amphotericin B, an antifungal agent, increased the intracellular platinum accumulation in resistant cells to the same level as in parent cells. Western blot analysis demonstrated that the Na+, K+-ATPase alpha1 subunit was reduced in resistant cells compared with the parental cells, although there was no difference in the expression of the beta1 subunit between the two cell lines. Furthermore, the Na+, K+-ATPase alpha1 subunit of H4-II-E was decreased following a 24-h exposure to CDDP. These results suggest that Na+, K+-ATPase-dependent active transport of CDDP does not occur in resistant cells, and, furthermore, our findings provide the first evidence that the Na+, K+-ATPase alpha1 subunit plays an important role in the transport of CDDP.
本研究旨在确定调节细胞内顺铂（CDDP）蓄积的因素以及CDDP耐药细胞系膜组分的变化。与亲代H4-II-E细胞相比，CDDP耐药大鼠肝癌细胞系H4-II-E/CDDP细胞内的铂蓄积显著减少，尽管这两种细胞系之间CDDP的外排没有差异。在本研究中，我们检测了主动转运功能变化对H4-II-E/CDDP细胞CDDP耐药性的影响。与耐药细胞相比，低温或ATP耗竭可使亲代细胞中的铂蓄积明显减少。此外，Na +，K + -ATP酶抑制剂哇巴因和K +通道抑制剂四乙铵可降低亲代细胞中的铂蓄积，但对耐药细胞中的蓄积没有影响。抗真菌剂两性霉素B可使耐药细胞内的铂蓄积增加至与亲代细胞相同的水平。蛋白质免疫印迹分析表明，与亲代细胞相比，耐药细胞中Na +，K + -ATP酶α1亚基减少，尽管两种细胞系之间β1亚基的表达没有差异。此外，H4-II-E细胞暴露于CDDP 24小时后，其Na +，K + -ATP酶α1亚基减少。这些结果表明，耐药细胞中不存在Na +，K + -ATP酶依赖性的CDDP主动转运，此外，我们的研究结果首次证明Na +，K + -ATP酶α1亚基在CDDP转运中起重要作用。
