Uptake of 3-[125I]iodo-α-methyl-l-tyrosine into colon cancer DLD-1 cells: characterization and inhibitory effect of natural amino acids and amino acid-like drugs☆
Received 20 June 2007; received in revised form 7 October 2009; accepted 31 October 2009. published online 30 November 2009.
Abstract
Introduction
We examined 3-[123I]iodo-α-methyl-l-tyrosine ([123I]IMT) uptake and inhibition by amino acids and amino acid-like drugs in the human DLD-1 colon cancer cell line, to discuss correlation between the inhibition effect and structure.
Methods
Expression of relevant neutral amino acid transporters was examined by real-time PCR with DLD-1 cells. The time course of [125I]IMT uptake, contributions of transport systems, concentration dependence and inhibition effects by amino acids and amino acid-like drugs (1 mM) on [125I]IMT uptake were examined.
Results
Expression of system L (4F2hc, LAT1 and LAT2), system A (ATA1, ATA2) and system ASC (ASCT1) was strongly detected; system L (LAT3, LAT4) and MCT8 were weakly detected; and B0AT was not detected. [125I]IMT uptake in DLD-1 cells involved Na+-independent system L primarily and Na+-dependent system(s). Uptake of [125I]IMT in Na+-free buffer followed Michaelis–Menten kinetics, with a Km of 78 μM and Vmax of 333 pmol/106 cells per minute. Neutral d- and l-amino acids with branched or aromatic large side chains inhibited [125I]IMT uptake. Tyrosine analogues, tryptophan analogues, l-phenylalanine and p-halogeno-l-phenylalanines, and gamma amino acids [including 3,4-dihydroxy-l-phenylalanine (l-DOPA), dl-threo-β-(3,4-dihydroxyphenyl)serine (DOPS), 4-[bis(2-chloroethyl)amino]-l-phenylalanine and 1-(aminomethyl)-cyclohexaneacetic acid] strongly inhibited [125I]IMT uptake, but l-tyrosine methyl ester and R(+)/S(−)-baclofen weakly inhibited uptake. The substrates of system ASC and A did not inhibit [125I]IMT uptake except l-serine and d/l-cysteine.
Conclusions
[125I]IMT uptake in DLD-1 cells involves mostly LAT1 and its substrates' (including amino acid-like drugs derived from tyrosine, tryptophan and phenylalanine) affinity to transport via LAT1. Whether transport of gamma amino acid analogues is involved in LAT1 depends on the structure of the group corresponding to the amino acid residue. Beta-hydroxylation may confer reduction of transport affinity of tyrosine analogues via LAT1.
☆ This work was supported by Grants-in-Aid for Scientific Research (#10770451, #14770498, #13557075, #15659283, #16659322 and #17390336) from the Ministry of Education, Science, Sports and Culture of Japan and the Japan Society for the Promotion of Science. Financial support was also provided by the Ibaraki Prefectural University Research Project (9808, 0118, and 0220); Ibaraki Prefectural University Grants-in-Aid of the Encouragement for Young Scientists 2001, 2002, 2004, 2005, 2006, 2008 and 2009; and Japan Atherosclerosis Research Foundation Grant-2008.
ABSTRACT