Adema Auke D, Floor Karijn, Smid Kees, Honeywell Richard J, Scheffer George L, Jansen Gerrit, Peters Godefridus J
Department of Medical Oncology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands.
Pathology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands.
Springerplus. 2014 Dec 13;3:732. doi: 10.1186/2193-1801-3-732. eCollection 2014.
We aimed to determine whether the multidrug-resistance-proteins MRP4 (ABCC4) and MRP5 (ABCC5) confer resistance to the antimetabolites cytarabine (Ara-C), gemcitabine (GEM), and the L-nucleoside analog troxacitabine. For this purpose we used HEK293 and the transfected HEK/MRP4 (59-fold increased MRP4) or HEK/MRP5i (991-fold increased MRP5) as model systems and tested the cells for drug sensitivity using a proliferation test. Drug accumulation was performed by using radioactive Ara-C, and for GEM and troxacitabine with HPLC with tandem-MS or UV detection. At 4-hr exposure HEK/MRP4 cells were 2-4-fold resistant to troxacitabine, ara-C and 9-(2-phosphonylmethoxyethyl)adenine (PMEA), and HEK/MRP5i to ara-C and PMEA, but none to GEM. The inhibitors probenecid and indomethacin reversed resistance. After 4-hr exposure ara-C-nucleotides were 2-3-fold lower in MRP4/5 cells, in which they decreased more rapidly after washing with drug-free medium (DFM). Trocacitabine accumulation was similar in the 3 cell lines, but after the DFM period troxacitabine decreased 2-4-fold faster in MRP4/5 cells. Troxacitabine-nucleotides were about 25% lower in MRP4/5 cells and decreased rapidly in MRP4, but not in MRP5 cells. Accumulation of GEM-nucleotides was higher in the MRP4/5 cells.
MRP4 and MRP5 overexpression confer resistance to troxacitabine and ara-C, but not to GEM, which was associated with a rapid decline of the ara-C and troxacitabine-nucleotides in HEK/MRP4-5 cells.
我们旨在确定多药耐药蛋白MRP4（ABCC4）和MRP5（ABCC5）是否赋予对抗代谢物阿糖胞苷（Ara - C）、吉西他滨（GEM）和L - 核苷类似物曲西他滨的耐药性。为此，我们使用HEK293以及转染的HEK/MRP4（MRP4增加59倍）或HEK/MRP5i（MRP5增加991倍）作为模型系统，并使用增殖试验测试细胞的药物敏感性。通过使用放射性阿糖胞苷进行药物蓄积，并通过高效液相色谱串联质谱或紫外检测对吉西他滨和曲西他滨进行检测。在4小时暴露时，HEK/MRP4细胞对曲西他滨、阿糖胞苷和9 - （2 - 膦酰甲氧基乙基）腺嘌呤（PMEA）有2至4倍的耐药性，HEK/MRP5i对阿糖胞苷和PMEA有耐药性，但对吉西他滨没有。抑制剂丙磺舒和吲哚美辛可逆转耐药性。在4小时暴露后，MRP4/5细胞中的阿糖胞苷核苷酸低2至3倍，在用无药培养基（DFM）洗涤后它们下降得更快。曲西他滨在这三种细胞系中的蓄积相似，但在DFM期后，曲西他滨在MRP4/5细胞中下降快2至4倍。MRP4/5细胞中的曲西他滨核苷酸低约25%，在MRP4细胞中迅速下降，但在MRP5细胞中不下降。MRP4/5细胞中吉西他滨核苷酸的蓄积更高。
MRP4和MRP5的过表达赋予对曲西他滨和阿糖胞苷的耐药性，但对吉西他滨没有，这与HEK/MRP4 - 5细胞中阿糖胞苷和曲西他滨核苷酸的快速下降有关。
