System L amino acid transporter inhibitor enhances anti-tumor activity of cisplatin in a head and neck squamous cell carcinoma cell line.

LAT1, a subunit of heterodimeric system L transporter responsible for transporting neutral amino acids into cells, has been investigated in several cancers because of its onco-fetal nature. Based on the studies of its functional inhibition, LAT1 has been proposed to be a new molecular target of a cancer therapy. We have shown here that human head and neck cancer cell line, Hep-2, expresses both LAT1 and 4F2hc, another subunit of system L transporter. An inhibitor of system L, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), inhibited leucine uptake by the cells. BCH administration or restriction of essential amino acid leucine decreased viability of Hep-2 cells. Co-administration of cisplatin with BCH reduced the viability of the cells more than either agent alone. When BCH treatment preceded cisplatin administration, reduction in Hep-2 cell viability was additive. In contrast, when BCH was given after cisplatin treatment, synergistic effect in decreasing the number of viable cells was obtained. BCH treatment decreased the phosphorylation of mTOR, p70S6K and 4EBP1, suggesting that BCH enhanced anti-tumor action of cisplatin by inhibiting mTOR pathway. This potentiation may be used to reduce cisplatin exposure to alleviate many unwanted toxicity of the drug.

Apical and basolateral 4F2hc and the amino acid exchange of L-DOPA in renal LLC-PK1 cells.

The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK1 cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [14C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [14C] L-DOPA handling. The basal-to-apical transepithelial flux of [14C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [14C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [14C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [14C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [14C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [14C] L-DOPA through the apical or the basal cell side. It is likely that the Na+-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.

Prevention of ornithine cytotoxicity by nonpolar side chain amino acids in retinal pigment epithelial cells.

PURPOSE: To investigate the effect of amino acids on ornithine cytotoxicity in ornithine-delta-aminotransferase (OAT)-deficient human retinal pigment epithelial (RPE) cells as an in vitro model of gyrate atrophy (GA) of the choroid and retina. METHODS: RPE cells were treated with 0.5 mM 5-fluoromethylornithine (5-FMOrn), a specific and irreversible OAT inhibitor. OAT-deficient RPE cells were incubated with 10 mM ornithine in the presence of 20 mM of 1 of 18 amino acids or 10 mM 2-amino-2-norbornane-carboxylic acid (BCH), a conventional inhibitor of the amino acid transporter system L. Ornithine cytotoxicity and cytoprotective effects of each amino acid was evaluated with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay 72 hours after treatment with ornithine in OAT-deficient RPE cells. Ornithine incorporation into RPE cells was evaluated using DL-[14C]ornithine. RESULTS: An MTT colorimetric assay revealed that small and large zwitterionic amino acids, but not acidic or basic amino acids, decreased ornithine cytotoxicity in OAT-deficient RPE cells. Incorporation of DL-[14C]ornithine by RPE cells decreased to 79% of the control level after incubation for 48 hours with 20 mM leucine, the most effective cytoprotective amino acid. Further, BCH prevented ornithine cytotoxicity in a dose-dependent manner. Both light and heavy chains of L-type amino acid transporter (LAT)-1, LAT2, y+LAT1, and 4F2hc were expressed in RPE cells. CONCLUSIONS: The present results demonstrate that L-type amino acid transporter(s) may be involved in protection against ornithine cytotoxicity in human RPE cells. Thus, amino acid transportation in RPE cells may be a good target for a new therapy for GA as well as other kinds of chorioretinal degeneration.