Department of Pharmacy, College of Chemical Engineering, Nanjing Forestry University, Nanjing 210000, China.
Department of Pharmaceutical Sciences, College of Pharmacy and Health Sciences, St. John's University, Queens, NY 11439, USA.
Int J Mol Sci. 2020 Feb 19;21(4):1387. doi: 10.3390/ijms21041387.
The overexpressing ABCB1 transporter is one of the key factors leading to multidrug resistance (MDR). Thus, many ABCB1 inhibitors have been found to be able to overcome ABCB1-mediated MDR. However, some inhibitors also work as a substrate of ABCB1, which indicates that in order to achieve an effective reversal dosage, a higher concentration is needed to overcome the pumped function of ABCB1, which may concurrently increase the toxicity. WYE-354 is an effective and specific mTOR (mammalian target of rapamycin) inhibitor, which recently has been reported to reverse ABCB1-mediated MDR. In the current study, 3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to determine the cell viability and reversal effect of WYE-354 in parental and drug-resistant cells. Drug accumulation was performed to examine the effect of WYE-354 on the cellular accumulation of chemotherapeutic drugs. The ATPase (adenosine triphosphatase) activity of the ABCB1 transporter in the presence or absence of WYE-354 was conducted in order to determine the impact of WYE-354 on ATP hydrolysis. Western blot analysis and immunofluorescence assay were used to investigate the protein molecules related to MDR. In addition, the interaction between the WYE-354 and ABCB1 transporter was investigated via in silico analysis. We demonstrated that WYE-354 is a substrate of ABCB1, that the overexpression of the ABCB1 transporter decreases the efficacy of WYE-354, and that the resistant WYE-354 can be reversed by an ABCB1 inhibitor at a pharmacological achievable concentration. Furthermore, WYE-354 increased the intracellular accumulation of paclitaxel in the ABCB1-mediated MDR cell line, without affecting the corresponding parental cell line, which indicated that WYE-354 could compete with other chemotherapeutic drugs for the ABCB1 transporter substrate binding site. In addition, WYE-354 received a high score in the docking analysis, indicating a strong interaction between WYE-354 and the ABCB1 transporter. The results of the ATPase analysis showed that WYE-354 could stimulate ABCB1 ATPase activity. Treatment with WYE-354 did not affect the protein expression or subcellular localization of the ABCB1. This study provides evidence that WYE-354 is a substrate of the ABCB1 transporter, implicating that WYE-354 should be avoided for use in ABCB1-mediated MDR cancer.
过度表达的 ABCB1 转运蛋白是导致多药耐药 (MDR) 的关键因素之一。因此，已经发现许多 ABCB1 抑制剂能够克服 ABCB1 介导的 MDR。然而，一些抑制剂本身也是 ABCB1 的底物，这表明为了达到有效的逆转剂量，需要更高的浓度来克服 ABCB1 的泵送功能，这可能会同时增加毒性。WYE-354 是一种有效的、特异性的 mTOR（哺乳动物雷帕霉素靶蛋白）抑制剂，最近有报道称其可逆转 ABCB1 介导的 MDR。在本研究中，采用 3-(4,5-二甲基噻唑基)-2,5-二苯基四氮唑溴盐（MTT）法测定亲本和耐药细胞中 WYE-354 的细胞活力和逆转作用。进行药物积累实验以检查 WYE-354 对化疗药物细胞内积累的影响。在存在或不存在 WYE-354 的情况下进行 ABCB1 转运蛋白的 ATP 酶（三磷酸腺苷酶）活性，以确定 WYE-354 对 ATP 水解的影响。通过 Western blot 分析和免疫荧光分析研究与 MDR 相关的蛋白分子。此外，通过计算机分析研究了 WYE-354 与 ABCB1 转运蛋白之间的相互作用。我们证明 WYE-354 是 ABCB1 的底物，ABCB1 转运蛋白的过度表达降低了 WYE-354 的功效，并且在药理学可达到的浓度下，ABCB1 抑制剂可逆转耐药的 WYE-354。此外，WYE-354 增加了 ABCB1 介导的 MDR 细胞系中紫杉醇的细胞内积累，而对相应的亲本细胞系没有影响，这表明 WYE-354 可以与其他化疗药物竞争 ABCB1 转运蛋白底物结合位点。此外，WYE-354 在对接分析中获得了高分，表明 WYE-354 与 ABCB1 转运蛋白之间存在强烈相互作用。ATP 酶分析结果表明，WYE-354 可以刺激 ABCB1 ATP 酶活性。用 WYE-354 处理不会影响 ABCB1 的蛋白表达或亚细胞定位。这项研究提供了证据表明 WYE-354 是 ABCB1 转运蛋白的底物，这意味着 WYE-354 不应在 ABCB1 介导的 MDR 癌症中使用。
