The blood–brain barrier is composed of both a physical barrier and an enzymatic barrier. Tight junction (TJ) proteins expressed between endothelial cells of brain capillaries provide the physical barrier to paracellular movement of ions and molecules to the brain, while luminal-facing efflux transporters enzymatically restrict the entry of blood-borne molecules from entering the brain. The expression and activity of ATP Binding Cassette transporters or “ABC” transporters in endothelial cells of the BBB and in human tumor cells are dynamically regulated by numerous signaling pathways. P-glycoprotein (P-gp), (ABCB1), is arguably the most studied transporter of the BBB, and in human cell lines. P-glycoprotein transport activity is rapidly inhibited by signaling pathways that call for the rapid production of nitric oxide (NO) from the inducible nitric oxide synthase enzyme, iNOS. This study investigated how nano-molar levels of the selective chemotherapeutic erastin affect the activity or expression of P-glycoprotein transporter in brain capillaries and in human tumor cell lines. We chose erastin because it signals to iNOS for NO production at low concentrations. Furthermore, erastin inhibits the cellular uptake of cystine through the XC−cystine/glutamate antiporter. Since previous reports indicate that NO production from iNOS can rapidly inhibit P-gp activity in tumor cells, we wondered if induction of iNOS by erastin could also rapidly reduce P-glycoprotein transport activity in brain endothelial cells and in human tumor cell lines. We show here that low concentrations of erastin (1 nM) can induce iNOS, inhibit the activity of P-glycoprotein, and reduce the intracellular uptake of cystine via the Xc- cystine/glutamate antiporter. Consistent with reduced P-glycoprotein activity in rat brain capillary endothelial cells, we show that human tumor cell lines exposed to erastin become more sensitive to cytotoxic substrates of P-glycoprotein.
血脑屏障由物理屏障和酶屏障共同构成。脑毛细血管内皮细胞间表达的紧密连接蛋白为离子和分子向大脑的旁细胞途径运动提供了物理屏障，而面向管腔的外排转运蛋白则通过酶促作用限制血源性分子进入大脑。血脑屏障内皮细胞及人类肿瘤细胞中ATP结合盒转运蛋白（即“ABC”转运蛋白）的表达与活性受到多种信号通路的动态调控。P-糖蛋白（ABCB1）堪称血脑屏障及人类细胞系中研究最为深入的转运蛋白。其转运活性可被诱导型一氧化氮合酶快速产生一氧化氮的信号通路迅速抑制。本研究探讨了纳摩尔级选择性化疗药物erastin如何影响脑毛细血管及人类肿瘤细胞系中P-糖蛋白转运体的活性与表达。选择erastin作为研究对象，是因为其在低浓度下即可通过信号传导激活诱导型一氧化氮合酶产生一氧化氮。此外，erastin能通过XC−胱氨酸/谷氨酸逆向转运蛋白抑制细胞对胱氨酸的摄取。鉴于既往研究表明诱导型一氧化氮合酶产生的一氧化氮可快速抑制肿瘤细胞中P-糖蛋白活性，我们推测erastin诱导的诱导型一氧化氮合酶是否同样能快速降低脑内皮细胞及人类肿瘤细胞系中P-糖蛋白的转运活性。本研究发现，低浓度erastin（1 nM）可诱导诱导型一氧化氮合酶表达、抑制P-糖蛋白活性，并通过Xc-胱氨酸/谷氨酸逆向转运蛋白降低细胞内胱氨酸摄取。与大鼠脑毛细血管内皮细胞中P-糖蛋白活性降低的现象一致，研究显示暴露于erastin的人类肿瘤细胞系对P-糖蛋白的细胞毒性底物敏感性增强。
A Role for iNOS in Erastin Mediated Reduction of P-Glycoprotein Transport Activity
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