Gastroenterology

Gastroenterology

Volume 120, Issue 7, June 2001, Pages 1689-1699
Gastroenterology

Alimentary Tract
LST-2, A human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers*,**,*,**,

https://doi.org/10.1053/gast.2001.24804Get rights and content

Abstract

Background & Aims: One approach to the development of targeted cancer chemotherapy exploits increased uptake of the agent into neoplastic cells. In this scenario, higher concentrations of the agent in cancer cells are responsible for differential killing, whereas the low concentration in normal human cells decreases side effects. The aim of this study was to isolate an organic anion transporter that is weak in normal cells, but abundantly expressed in cancer cells, to deliver the anticancer drugs to the cells. Methods: A human liver complementary DNA (cDNA) library was screened with liver-specific transporter (LST)-1 cDNA as a probe. Northern blot analyses were performed using the isolated cDNA (termed LST-2). An LST-2–specific antibody was raised, and immunohistochemical analyses including immunoelectron microscopy were performed. Xenopus oocyte expression system was used for functional analysis. We also established a permanent cell line that consistently expresses LST-2 to examine the relationship between methotrexate uptake and sensitivity. Results: The isolated cDNA, LST-2, has 79.7% of overall homology with human LST-1. LST-2 exclusively expressed in the liver under normal conditions and its immunoreactivity was highest at the basolateral membrane of the hepatocytes around the central vein. Although its weak expression in the liver, LST-2 is abundantly expressed in the gastric, colon, and pancreatic cancers. On the other hand, the LST-1 was only detected in a hepatic cell line. LST-2 transports methotrexate in a saturable and dose-dependent manner. Furthermore, introduction of the LST-2 gene into mammalian cells potentiates sensitivity to methotrexate. Conclusions: LST-2 is one of the prime candidate molecules for determining methotrexate sensitivity and may be a good target to deliver anticancer drugs to the gastrointestinal cancers.

GASTROENTEROLOGY 2001;120:1689-1699

Section snippets

Isolation of the human liver-specific transporter 2 cDNA

A human liver complementary DNA (cDNA) library was constructed using the λZAPII vector (Stratagene).10 Independent clones (5 × 105) were screened with human liver-specific transporter (LST)-111 under low stringency. As a result, 42 independent hybridization-positive clones were isolated. Among these, 1 clone (pH 7), which was distinct from LST-1, was isolated. Multiple sequence alignments of amino acid sequences and phylogenetic tree construction were carried out using Clustal W.12 The

Isolation and hepatic distribution

The isolated cDNA, termed LST-2, encodes 702 amino acids (Mr 77,398). The hydrophobicity analysis20 suggests the presence of 12 transmembrane domains, which is characteristic of organic anion transporters (Figure 1A).

. Sequence alignment and analysis of LST-2. (A) Amino acid alignment of LST-1 and LST-2 (GenBank accession no. AF187815). The sequences are aligned with single-letter notation by inserting gaps (–) to achieve the maximum homology. Exact matches and conservative substitutions are

Conclusion

We have shown that LST-2 is one of a key molecule in the delivery of MTX to gastrointestinal cancers. Because of the exclusive expression of LST-2 in the cancer cells, LST-2 may represent a new therapeutic target for the treatment of such cancers with less side effects.

Acknowledgements

The authors thank Drs. K. J. Seth and M. R. Kaplan for discussions.

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*

Address requests for reprints to: Takaaki Abe, Division of Nephrology, Endocrinology, and Vascular Medicine, Department of Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-cho, Aobaku, Sendai, 980-8574, Japan. e-mail: [email protected]; fax: (81) 22-717-7168.

**

Drs. Abe, Unno, and Onogawa contributed equally to this study.

*

Supported in part by research grants from the Ministry of Education, Science and Culture of Japan, Yamanouchi Foundation, Osaka Cancer Research Foundation, Nakatomi Foundation, Novartis Foundation, Ono Medical Research Foundation, Inamori Foundation, Welfide Medical Research Foundation, and Uehara Memorial Foundation.

**

This study was approved by the Ethical Committee of Tohoku University.

The sequence of human LST-2 has been deposited under the GenBank accession number AF187815, and the genomic sequence of LST-2 has been deposited under the Genbank accession number AF195785.

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