Hooijberg Jan Hendrik, Jansen Gerrit, Assaraf Yehuda G, Kathmann Ietje, Pieters Rob, Laan Adrie C, Veerman Anjo J P, Kaspers Gertjan J L, Peters Godefridus J
Department of Pediatric Hematology/Oncology, VU University Medical Center (VUMC), De Boelelaan 1117, 1007 MB, Amsterdam, The Netherlands.
Biochem Pharmacol. 2004 Apr 15;67(8):1541-8. doi: 10.1016/j.bcp.2003.12.022.
The Multidrug Resistance Protein MRP1 (ABCC1) can confer resistance to a variety of therapeutic drugs. In addition, MRP1/ABCC1 mediates cellular export of natural folates, such as folic acid and l-leucovorin. In this study we determined whether cellular folate status affected the functional activity of MRP1/ABCC1 mediated efflux of an established substrate, the anthracycline daunorubicin (DNR). As a model system we used the human ovarian carcinoma cell line 2008wt, and its MRP1/ABCC1 transfected subline 2008/MRP1. Both types of these moderate- and high-MRP1/ABCC1 expressing cells displayed efflux of DNR when maintained in standard culture media (2.3microM folic acid). The initial total cellular DNR efflux rate in 2008/MRP1 cells was approximately 2-fold higher compared to 2008wt cells. This efflux consisted of MRP1/ABCC1 mediated transport, possibly non-MRP1 mediated transport, as well as passive diffusion. Benzbromarone, a specific MRP1 inhibitor, decreased the initial efflux rate in 2008/MRP1 cells (4-fold) and in 2008wt cells (2-fold). When 2008/MRP1 cells were challenged for 2 days in folate-free medium, total cellular DNR efflux was decreased to 43% of the initial efflux rate under folate-rich conditions. In 2008wt cells DNR efflux was decreased to 84% of the folate-rich conditions. Benzbromarone did not inhibit DNR efflux after the folate-free period in both cell lines. Repletion of folate by a 2-24hr exposure to 2.5microM l-leucovorin or folic acid resulted in a complete restoration of DNR efflux. In contrast, expression of MRP1/ABCC1 protein was not changed significantly during the folate-free period or the repletion-period, nor were cellular ATP or ADP pools. In conclusion, this study demonstrates that the cellular folate status can influence the transport activity of MRP1/ABCC1. These results have potentially important implications in the understanding of the (patho-)physiological roles of MRP1/ABCC1, and possibly other ABC transporter proteins in cellular folate homeostasis and drug resistance.
多药耐药蛋白MRP1（ABCC1）可赋予对多种治疗药物的耐药性。此外，MRP1/ABCC1介导天然叶酸（如叶酸和左亚叶酸）的细胞外排。在本研究中，我们确定细胞叶酸状态是否会影响MRP1/ABCC1介导的既定底物柔红霉素（DNR）外排的功能活性。作为模型系统，我们使用了人卵巢癌细胞系2008wt及其MRP1/ABCC1转染亚系2008/MRP1。当维持在标准培养基（2.3μM叶酸）中时，这两种中等和高表达MRP1/ABCC1的细胞类型均表现出DNR外排。2008/MRP1细胞中最初的总细胞DNR外排率比2008wt细胞高约2倍。这种外排包括MRP1/ABCC1介导的转运、可能的非MRP1介导的转运以及被动扩散。特异性MRP1抑制剂苯溴马隆降低了2008/MRP1细胞（4倍）和2008wt细胞（2倍）中的初始外排率。当2008/MRP1细胞在无叶酸培养基中培养2天时，总细胞DNR外排降至富含叶酸条件下初始外排率的43%。在2008wt细胞中，DNR外排降至富含叶酸条件下的84%。在两个细胞系无叶酸培养期后，苯溴马隆均不抑制DNR外排。通过2 - 24小时暴露于2.5μM左亚叶酸或叶酸来补充叶酸，可使DNR外排完全恢复。相比之下，在无叶酸期或补充期，MRP1/ABCC1蛋白的表达没有显著变化，细胞ATP或ADP池也没有变化。总之，本研究表明细胞叶酸状态可影响MRP1/ABCC1的转运活性。这些结果对于理解MRP1/ABCC1以及可能其他ABC转运蛋白在细胞叶酸稳态和耐药性中的（病理）生理作用具有潜在的重要意义。
