Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner.
Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner.
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DOI：
10.1371/journal.pone.0186924
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Yue W
中科院分区：
文献类型：
--
作者：
Alam K;Farasyn T;Crowe A;Ding K;Yue W
OATP1B1和OATP1B3介导肝脏对多种药物(如他汀类药物)的摄取，并可介导转运体介导的药物-药物相互作用(DDIS)。Bortezomib是美国食品和药物管理局批准的治疗多发性骨髓瘤的一类蛋白酶体抑制剂药物。Bortezomib引起OATP介导的DDIS的可能性尚未得到评估。本研究探讨了泛素-蛋白酶体系统(UPS)在OATP1B1和OATP1B3降解中的作用，并确定了蛋白酶体抑制剂对OATP1B1和OATP1B3介导的转运的影响。在FLAG标记的OATP1B1/OATP1B3和血凝素标记的泛素(HA)标记的泛素共转染人胚胎肾(HEK)293细胞中，观察到FLAG-OATP1B1/1B3和HA-泛素的免疫共沉淀，表明OATP1B1和OATP1B3可以被泛素修饰。虽然在HEK293-FLAG-OATP1B1和-OATP1B3细胞中，用硼替佐米(50 nM，7h)阻断蛋白酶体活性增加了内源性泛素结合的FLAG-OATP1B1和FLAG-OATP1B3，但这种处理并不影响OATP1B1和OATP1B3的总蛋白水平，这表明在当前的构造条件下，UPS在OATP1B1和OATP1B3的降解中起到的作用较小。Bortezomib(50-250 nm，2-7 h)可显著降低HEK293-OATP1B3和人肝细胞(SCH)对OATP1B3底物[~3H]CCK-8的转运。然而，在HEK293-OATP1B1/1B3细胞和/或人SCH细胞中，硼替佐米对OATP1B1和OATP1B3的双重底物[~3H]E217βG和[~3H]Pitavastatin的转运几乎没有影响。与载体对照组相比，Bortezomib预处理显著降低了OATP1B3介导的CCK-8的最大转运速度(Vmax)(92.25±14.2vs.133.95±15.5pmol/mg蛋白/分钟)，而不影响亲和常数(Km)。其他蛋白酶体抑制剂MG132、环氧米星和卡菲佐米也显著降低了OATP1B3介导的[~3H]CCK-8转运。综上所述，目前的研究首次报道了OATP1B1和OATP1B3的泛素化以及Bortezomib对OATP1B3介导的转运的明显底物依赖的抑制作用。这些数据表明，Bortezomib引起OATP介导的DDIS的风险很低。
OATP1B1 and OATP1B3 mediate hepatic uptake of many drugs (e.g., statins) and can mediate transporter-mediated drug-drug-interactions (DDIs). Bortezomib is the first-in-class proteasome inhibitor drug approved by the U. S. Food and Drug Administration for the treatment of multiple myeloma. The potential of bortezomib to cause OATP-mediated DDIs has not been assessed. The current study investigated the involvement of the ubiquitin-proteasome system (UPS) in OATP1B1 and OATP1B3 degradation and determined the effects of proteasome inhibitors on OATP1B1- and OATP1B3-mediated transport. Co-immunoprecipitation of FLAG-OATP1B1/1B3 and HA-ubiquitin was observed in human embryonic kidney (HEK) 293 cells co-transfected with FLAG-tagged OATP1B1/OATP1B3 and hemagglutinin (HA)-tagged ubiquitin, suggesting that OATP1B1 and OATP1B3 can be ubiquitin-modified. Although blocking proteasome activity by bortezomib treatment (50 nM, 7 h) increased the endogenous ubiquitin-conjugated FLAG-OATP1B1 and FLAG-OATP1B3 in HEK293-FLAG-OATP1B1 and–OATP1B3 cells, such treatment did not affect the total protein levels of OATP1B1 and OATP1B3, suggesting that the UPS plays a minor role in degradation of OATP1B1 and OATP1B3 under current constitutive conditions. Pretreatment with bortezomib (50–250 nM, 2–7 h) significantly decreased transport of [3H]CCK-8, a specific OATP1B3 substrate, in HEK293-OATP1B3 and human sandwich-cultured hepatocytes (SCH). However, bortezomib pretreatment had negligible effects on the transport of [3H]E217βG and [3H]pitavastatin, dual substrates of OATP1B1 and OATP1B3, in HEK293-OATP1B1/1B3 cells and/or human SCH. Compared with vehicle control treatment, bortezomib pretreatment significantly decreased the maximal transport velocity (Vmax) of OATP1B3-mediated transport of CCK-8 (92.25 ± 14.2 vs. 133.95 ± 15.5 pmol/mg protein/min) without affecting the affinity constant (Km) values. Treatment with other proteasome inhibitors MG132, epoxomicin, and carfilzomib also significantly decreased OATP1B3-mediated [3H]CCK-8 transport. In summary, the current studies for the first time report ubiquitination of OATP1B1 and OATP1B3 and the apparent substrate-dependent inhibitory effect of bortezomib on OATP1B3-mediated transport. The data suggest that bortezomib has a low risk of causing OATP-mediated DDIs.