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Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner.

ABSTRACT: 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]E217?G 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.

SUBMITTER: Alam K

PROVIDER: S-EPMC5673231 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner.

Alam Khondoker K Farasyn Taleah T Crowe Alexandra A Ding Kai K Yue Wei W

PloS one 20171106 11

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 dete ...[more]

PMID: 29107984

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Project description:OBJECTIVE:Recent innovations in biology are boosting gene and cell therapy, but monitoring the response to these treatments is difficult. The purpose of this study was to find an MRI-reporter gene that can be used to monitor gene or cell therapy and that can be delivered without a viral vector, as viral vector delivery methods can result in long-term complications. MATERIALS AND METHODS:CMV promoter-human organic anion transporting polypeptide 1B3 (CMV-hOATP1B3) cDNA or CMV-blank DNA (control) was transfected into HEK293 cells using Lipofectamine. OATP1B3 expression was confirmed by western blotting and confocal microscopy. In vitro cell phantoms were made using transfected HEK293 cells cultured in various concentrations of gadoxetic acid for 24 hours, and images of the phantoms were made with a 9.4T micro-MRI. In vivo xenograft tumors were made by implanting HEK293 cells transfected with CMV-hOATP1B3 (n = 4) or CMV-blank (n = 4) in 8-week-old male nude mice, and MRI was performed before and after intravenous injection of gadoxetic acid (1.2 ?L/g). RESULTS:Western blot and confocal microscopy after immunofluorescence staining revealed that only CMV-hOATP1B3-transfected HEK293 cells produced abundant OATP1B3, which localized at the cell membrane. OATP1B3 expression levels remained high through the 25th subculture cycle, but decreased substantially by the 50th subculture cycle. MRI of cell phantoms showed that only the CMV-hOATP1B3-transfected cells produced a significant contrast enhancement effect. In vivo MRI of xenograft tumors revealed that only CMV-hOATP1B3-transfected HEK293 tumors demonstrated a T1 contrast effect, which lasted for at least 5 hours. CONCLUSION:The human endogenous OATP1B3 gene can be non-virally delivered into cells to induce transient OATP1B3 expression, leading to gadoxetic acid-mediated enhancement on MRI. These results indicate that hOATP1B3 can serve as an MRI-reporter gene while minimizing the risk of long-term complications.

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Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner.

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Treatment with proteasome inhibitor bortezomib decreases organic anion transporting polypeptide (OATP) 1B3-mediated transport in a substrate-dependent manner.

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