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Reporter PET Images Bortezomib Treatment-Mediated Suppression of Cancer Cell Proteasome Activity.

ABSTRACT: Proteasomal protein degradation is a promising target for cancer therapy. Here, we developed a positron emission tomography (PET) technique based on the sodium-iodide symporter (NIS) gene fused with the carboxyl-terminal of ornithine decarboxylase (cODC) that noninvasively images cancer cells with inhibited proteasome activity. A retroviral vector was constructed in which the murine cODC degron was fused to the human NIS gene (NIS-cODC). Transiently transduced CT26 and HT29 colon cancer cells and stably expressing CT26/NIS-cODC cells were prepared. In cancer cells transiently transduced with NIS-cODC, NIS expression and transport activity was low at baseline, but NIS protein and 125I uptake was significantly increased by inhibition of proteasome activity with bortezomib. Stable CT26/NIS-cODC cells also showed increased cytosolic and membrane NIS by bortezomib, and four different stable clones displayed bortezomib dose-dependent stimulation of 125I and 99mTc-04- uptake. Importantly, bortezomib dose-dependently suppressed survival of CT26/NIS-cODC clones in a manner that closely correlated to the magnitudes of 125I and 99mTc-04- uptake. CT26/NIS-cODC tumors of bortezomib-treated mice demonstrated greater 124I uptake on PET images and increased NIS expression on tissue staining compared to vehicle-injected animals. NIS-cODC PET imaging may allow noninvasive quantitative monitoring of proteasome activity in cancer cells treated with bortezomib.

SUBMITTER: Lee JH

PROVIDER: S-EPMC6095884 | biostudies-other | 2018 Aug

REPOSITORIES: biostudies-other

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Reporter PET Images Bortezomib Treatment-Mediated Suppression of Cancer Cell Proteasome Activity.

Lee Jin Hee JH Jung Kyung-Ho KH Quach Cung Hoa Thien CHT Park Jin Won JW Moon Seung Hwan SH Cho Young Seok YS Lee Kyung-Han KH

Scientific reports 20180816 1

Proteasomal protein degradation is a promising target for cancer therapy. Here, we developed a positron emission tomography (PET) technique based on the sodium-iodide symporter (NIS) gene fused with the carboxyl-terminal of ornithine decarboxylase (cODC) that noninvasively images cancer cells with inhibited proteasome activity. A retroviral vector was constructed in which the murine cODC degron was fused to the human NIS gene (NIS-cODC). Transiently transduced CT26 and HT29 colon cancer cells an ...[more]

PMID: 30116045

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Project description: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.

Project description:BackgroundMuscle wasting, a prominent feature of cancer cachexia, is mainly caused by sustained protein hypercatabolism. The enhanced muscle protein degradation rates rely on the activity of different proteolytic systems, although the Adenosine triphosphate (ATP)-ubiquitin-proteasome-dependent pathway and autophagy have been shown to play a pivotal role. Bortezomib is a potent reversible and selective proteasome and NF-κB inhibitor approved for the clinical use, which has been shown to be effective in preventing muscle wasting in different catabolic conditions. The aim of the present study has been to investigate whether pharmacological inhibition of proteasome by bortezomib may prevent skeletal muscle wasting in experimental cancer cachexia.MethodsCancer cachexia was induced in rats by intraperitoneal injection of Yoshida AH-130 ascites hepatoma cells and in mice by subcutaneous inoculation of C26 carcinoma cells. Animals were then further randomized to receive bortezomib. The AH-130 hosts were weighted and sacrificed under anaesthesia, on Days 3, 4, 5, and 7 after tumour inoculation, while C26-bearing mice were weighted and sacrificed under anaesthesia 12 days after tumour transplantation. NF-κB and proteasome activation, MuRF1 and atrogin-1 mRNA expression and beclin-1 protein levels were evaluated in the gastrocnemius of controls and AH-130 hosts.ResultsBortezomib administration in the AH-130 hosts, although able to reduce proteasome and NF-κB DNA-binding activity in the skeletal muscle on Day 7 after tumour transplantation, did not prevent body weight loss and muscle wasting. In addition, bortezomib exerted a transient toxicity, as evidenced by the reduced food intake and by the increase in NF-κB DNA-binding activity in the AH-130 hosts 3 days after tumour transplantation. Beclin-1 protein levels were increased by bortezomib treatment in Day 3 controls but were unchanged on both Days 3 and 7 in the AH-130 hosts, suggesting that an early compensatory induction of autophagy may exist in healthy but not in tumour-bearing animals. Regarding C26-bearing mice, bortezomib did not prevent as well body and muscle weight loss 12 days after tumour implantation.ConclusionsThe results obtained suggest that proteasome inhibition by bortezomib is not able to prevent muscle wasting in experimental cancer cachexia. Further studies are needed to address the issue whether a different dosage of bortezomib alone or in combination with other drugs modulating different molecular pathways may effectively prevent muscle wasting during cancer cachexia.

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Reporter PET Images Bortezomib Treatment-Mediated Suppression of Cancer Cell Proteasome Activity.

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Reporter PET Images Bortezomib Treatment-Mediated Suppression of Cancer Cell Proteasome Activity.

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