Cancer Letters

Cancer Letters

Volume 260, Issues 1–2, 18 February 2008, Pages 163-169
Cancer Letters

Rapid screening of antineoplastic candidates for the human organic anion transporter OATP1B3 substrates using fluorescent probes

https://doi.org/10.1016/j.canlet.2007.10.040Get rights and content

Abstract

A rapid screening system has been established to extract novel candidates that exhibit potent inhibition of the transport of fluorescent substrate by organic anion transporting polypeptide (OATP) 1B3. OATP1B3 is abundantly expressed in solid digestive organ cancers. Thus, the identification of new substrates leads to novel strategies for effective cancer chemotherapy with minimal adverse effects. We used an automated image acquisition and analysis system (IN Cell Analyzer 1000) to visualize the transport and subsequent accumulation of the fluorescent substrate chenodeoxycholyl-(Nε-NBD)-lysine (CDCA-NBD). Antineoplastic screening demonstrated that five candidates agents, docetaxel, actinomycin D, mitoxantrone, paclitaxel, and SN-38, exhibited potent inhibitory effects on OATP1B3-mediated transport of CDCA-NBD. To clarify if these antineoplastic drugs are substrates for OATP1B3, we performed transport assays in OATP1B3-expressing cells. We determined that SN-38 is a novel substrate for OATP1B3. In conclusion, our results demonstrate that the screening system established in this study is a useful method for the rapid extraction of candidate therapeutic agents from the large numbers of compounds.

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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