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☆

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 K_m of 78 μM and V_max 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.

Keywords: Amino acid-like drug, Colon cancer cell line, DLD-1, 3-Iodo-α-methyl-l-tyrosine, Inhibitor, l-Type amino acid tranasporter-1