Lemos Clara, Kathmann Ietje, Giovannetti Elisa, Dekker Henk, Scheffer George L, Calhau Conceição, Jansen Gerrit, Peters Godefridus J
Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, Porto, Portugal.
Int J Cancer. 2008 Oct 1;123(7):1712-20. doi: 10.1002/ijc.23677.
Folates can induce the expression and activity of the breast-cancer-resistance-protein (BCRP) and the multidrug-resistance-protein-1 (MRP1). Our aim was to study the time-dependent effect of folate deprivation/supplementation on (i) BCRP and MRP expression and (ii) on drug resistance mediated by these transporters. Therefore Caco-2 colon cancer cells usually grown in standard RPMI-medium containing supraphysiological folic acid (FA) concentrations (2.3 muM; high-folate, HF) were gradually adapted to more physiological folate concentrations (1 nM leucovorin (LV) or 1 nM FA; low-folate, LF), resulting in the sublines Caco-2-LF/LV and Caco-2-LF/FA. Caco-2-LF/LV and LF/FA cells exhibited a maximal increase of 5.2- and 9.6-fold for BCRP-mRNA and 3.9- and 5.7-fold for BCRP protein expression, respectively, but no major changes on MRP expression. Overexpression of BCRP in the LF-cells resulted in 3.6- to 6.3-fold resistance to mitoxantrone (MR), which was completely reverted by the BCRP inhibitor Ko143. On the other hand, LF-adapted cells were markedly more sensitive to methotrexate than the HF-counterpart, both after 4-hr (9,870- and 23,923-fold for Caco-2-LF/LV and LF/FA, respectively) and 72-hr (11- and 22-fold for Caco-2-LF/LV and LF/FA, respectively) exposure. Immunofluorescent staining observed with a confocal-laser-scan-microscope revealed that in Caco-2 cells (both HF and LF), BCRP is mainly located in the cytoplasm. In conclusion, folate deprivation induces BCRP expression associated with MR resistance in Caco-2 cells. The intracellular localization of BCRP in these cells suggests that this transporter is not primarily extruding its substrates out of the cell, but rather to an intracellular compartment where folates can be kept as storage.
叶酸可诱导乳腺癌耐药蛋白（BCRP）和多药耐药蛋白1（MRP1）的表达及活性。我们的目的是研究叶酸剥夺/补充对（i）BCRP和MRP表达以及（ii）由这些转运蛋白介导的耐药性的时间依赖性影响。因此，通常在含有超生理叶酸（FA）浓度（2.3 μM；高叶酸，HF）的标准RPMI培养基中生长的Caco-2结肠癌细胞逐渐适应更生理的叶酸浓度（1 nM亚叶酸（LV）或1 nM FA；低叶酸，LF），从而产生亚系Caco-2-LF/LV和Caco-2-LF/FA。Caco-2-LF/LV和LF/FA细胞的BCRP mRNA分别最大增加5.2倍和9.6倍，BCRP蛋白表达分别增加3.9倍和5.7倍，但MRP表达无重大变化。LF细胞中BCRP的过表达导致对米托蒽醌（MR）产生3.6至6.3倍的耐药性，BCRP抑制剂Ko143可完全逆转这种耐药性。另一方面，无论是在4小时（Caco-2-LF/LV和LF/FA分别为9870倍和23923倍）还是72小时（Caco-2-LF/LV和LF/FA分别为11倍和22倍）暴露后，LF适应细胞对甲氨蝶呤的敏感性均明显高于HF对应细胞。用共聚焦激光扫描显微镜观察的免疫荧光染色显示，在Caco-2细胞（HF和LF）中，BCRP主要位于细胞质中。总之，叶酸剥夺诱导Caco-2细胞中与MR耐药相关的BCRP表达。BCRP在这些细胞中的细胞内定位表明，该转运蛋白并非主要将其底物排出细胞，而是转运至细胞内隔室，叶酸可在其中储存。
