Rapid screening of antineoplastic candidates for the human organic anion transporter OATP1B3 substrates using fluorescent probes
Introduction
Organic anion transporting polypeptides (OATPs) are sodium-independent organic anion transporters expressed in a wide variety of tissues, including the liver, kidney, intestine, and brain. OATPs contribute to the transport of bile acids, thyroid hormones, steroid conjugates, anionic oligopeptides, eicosanoids, drugs, and other xenobiotic compounds across membranes [1], [2], [3], [4]. OATP1B3 (LST-2/OATP8), a member of the liver-specific subfamily of OATPs, localizes to the basolateral membrane of hepatocytes [5], [6], [7], [8]. OATP1B3 is also expressed by solid digestive organ neoplasms, including gastric, pancreatic, and colon cancers [7]. OATP1B3 is weakly expressed by the normal liver, but strongly upregulated by cancer cells. Thus, a greater understanding of the interaction between OATP1B3 and antineoplastic drugs would be useful to develop novel strategies for effective cancer chemotherapeutics with minimal adverse effects.
Fluorescent bile acids are efficiently transported by both OATP1B1 and OATP1B3 [9]. We have recently succeeded in the visualization of the transport process using confocal imaging. We applied this imaging technique to the screening of transporter substrates. In this study, we examined the effects of antineoplastic drugs on the transport of fluorescent substrates via OATP1B3 using an automated image acquisition and analysis system (IN Cell Analyzer 1000). We have discovered a new substrate for OATP1B3, suggesting that this system can be instrumental in the rapid screening of novel candidate substrates.
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Materials
Chenodeoxycholyl-(Nε-NBD)-lysine (CDCA-NBD) was synthesized as previously described [9]. Anti-OATP8 antibody was purchased from Affinity BioReagents, Inc. (Golden, CO). All other chemicals were commercially available and of the highest purity possible.
Cell culture and transfection studies
HEK293 cells, which are derived from human embryonic kidneys, were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum in an atmosphere of 5% CO2, 95% air at 37 °C. Cells were transfected with a pcDNA3.1(+)
Transport of CDCA-NBD by OATP1B3-transfected cells
First, we established stable clones overproducing OATP1B3. Clones that survived selection in 0.5 mg/mL G418 were expanded. Analysis for OATP1B3 overproduction by immunoblotting and CDCA-NBD uptake (Fig. 1A and B) isolated two clones, OATP1B3-9 and OATP1B3-10. OATP1B3-9 exhibited higher expression levels and transport activity of OATP1B3 than OATP1B3-10 (Fig. 1B). Kinetic parameters in OATP1B3-9 were determined after exposure to varying concentrations of CDCA-NBD (Fig. 1C). The apparent
Discussion
In this study, we developed a screening system to extract antineoplastic candidates that are OATP1B3 substrates using an automated image acquisition and analysis system (IN Cell Analyzer 1000). As OATP1B3 is abundantly expressed in liver and other solid digestive organ cancers [7], effective cancer chemotherapy could exploit the OATP1B3 protein to allow the increased accumulation of antineoplastic drugs in cancer cells.
CDCA-NBD is a fluorescent probe that is efficiently transported by OATP1B3 (
Acknowledgments
This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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