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.

Introduction

3-[¹²³I]Iodo-α-methyl-l-tyrosine ([¹²³I]IMT), an artificial amino acid, has been developed as a functional imaging agent for tyrosine transport mechanisms in the brain and pancreas [1], [2], [3]. Because amino acids rapidly accumulate in tumor cells for active proliferation, [¹²³I]IMT has also been used clinically for SPECT imaging of tumors, as described by Langen et al. [4], [5], [6], [7], [8].

Compared to intracranial tumor cell lines including rat C6 glioma cells [9], human GOS3 glioma cells [10] and 86HG-39 human glioma cells [8], relatively few of the studies of [¹²³I]IMT transport have involved extracranial tumor cell lines. The kinetics of [¹²³I]IMT transport have been studied in human Ewing's sarcoma cells [11]. Furthermore, [¹²³I]IMT transport has been characterized in rat lymphoma cells [12], a porcine kidney epithelial cell line [13], human monocyte-macrophages [14], human small-cell lung cancer [15] and pancreatic carcinoma [16].

In cultured glioma cells of the lines mentioned above, membrane transport of [¹²³I]IMT is dominated by BCH-sensitive transport, i.e., amino acid transport system L, and relatively minor uptake occurs via the Na⁺-dependent system [8], [9], [10]. System T also mediates [¹²³I]IMT transport into U266 human myeloma cells [17]. However, the gene expression of neutral amino acid transporters has not been fully clarified even in those cell lines.

Some of the system L transporters demonstrate gene expression inducement that is dependent on the cellular conditions, and thus researchers in the fields of cell activation, cellular proliferation and cellular nutrition have become interested in the inhibitors and/or substrates of the system L transporter family [18], [19], [20]. The results of such studies are applied to not only cancer imaging [7] or therapeutics [21], but also to the development of a new class of target in immunosuppressant studies [22].

In the present study, we characterized [¹²⁵I]IMT transport in the human colon cancer cell line, DLD-1, with particular emphasis on the inhibitory effect of amino acid-like drugs. The gene expression of neutral amino acid transporters was confirmed with real-time reverse transcription–PCR (real-time PCR) in DLD-1. We selected the following amino acid-like drugs for investigation: dl-threo-β-(3,4-dihydroxyphenyl)serine [DOPS; amino acid precursor of noradrenaline (NA); elevates brain NA concentrations] [23], 4-[bis(2-chloroethyl)amino]-l-phenylalanine (melphalan; antineoplastic agent that forms DNA intrastrand crosslinks by bifunctional alkylation in 5′-GGC sequences) [21], β-(aminomethyl)4-chlorobenzenepropanoic acid [R(+)-baclofen; GABAB receptor agonist: more active enantiomer; skeletal muscle relaxant, S(−)-baclofen and less active enantiomer of baclofen] [24], 1-(aminomethyl)-cyclohexaneacetic acid (gabapentin; anticonvulsant with unknown mechanism of action, crosses the blood–brain barrier, increases GABA concentrations in the brain and reduces excitatory amino acid neurotransmission) [25], and others as listed in Materials and Methods. In addition, we discuss the correlation between the inhibition effect and the structure of these compounds. To the best of our knowledge, there are no previously published data on the inhibitory effects of such amino acid analogues on [¹²⁵I]IMT transport.