Int J Pharm. 2013 Feb ;443(1-2): 245-253

Functional characterization and molecular expression of large neutral amino acid transporter (LAT1) in human prostate cancer cells.

Mitesh Patel1, Pranjali Dalvi2, Mitan Gokulgandhi1, Susamita Kesh3, Tanvi Kohli3, Ashim K. Mitra1*

1Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108, USA. 2Department of Internal Medicine, University of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, Kansas 66160, USA. 3School of Medicine, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108, USA.

 

ABSTRACT:

The primary objective of this study was to functionally characterize and provide molecular evidence of large neutral amino acid transporter (LAT1) in human derived prostate cancer cells (PC-3). We carried out the uptake of [3H]-tyrosine to delineate the functional activity of LAT1 transporter. Reverse transcription-polymerase chain reaction (RT-PCR) analysis was conducted to confirm the molecular expression of LAT1. [3H]-tyrosine uptake was found to be time dependent and linear up to 60 min. The uptake process did not exhibit any dependence on sodium ions, pH and energy. However, it was temperature dependent and found to be maximal at physiological temperature. [3H]-tyrosine uptake demonstrated saturable kinetics with Km and Vmax values of 34 ± 3 μM and 0.70 ± 0.02 nanomoles/min/mg protein, respectively. It was strongly inhibited by large neutral (phenylalanine, tryptophan, leucine, isoleucine) and small neutral (alanine, serine, cysteine) but not by basic (lysine and arginine) and acidic (aspartic and glutamic acid) amino acids. Amino acid modified quinidine, i.e., isoleucine–quinidine (Ile-quinidine) prodrug generated a two-fold higher uptake relative to unmodified quinidine. RT-PCR analysis provided a product band at 658 and 840 bp, specific to LAT1 and LAT2, respectively. In summary, this study provides the functional characterization and molecular evidence of LAT1 in prostate cancer cells.

PMID: 23270998

 

SUPPLEMENT:

System L, a heterodimer transporter contains 4F2hc heavy chain with LAT1 and LAT2 light chains. LAT1 transporter is principally responsible for the transport of large neutral amino acid with branched or aromatic side chains. LAT1 expression has already been demonstrated in intestine, kidney, placenta, retina and cornea. However, there are no previous reports on the functional characterization and molecular expression of LAT1 in human prostate cancer (PC-3) cells, PC-3 cells. We selected PC-3 cells, since these cells are considered an excellent in vitro cell culture model in human prostatic cancer. LAT1 transporter was functionally characterized using tyrosine as a model substrate.

Tyrosine uptake process appeared to be time dependent and linear up to 60 min. Moreover the uptake process appeared to be pH and Na+ independent. However, the process demonstrated high temperature dependency. The energy of activation obtained was found to be 4.8 kcal/mol. Tyrosine uptake remained unchanged in the presence of ouabain, 2,4-dinitrophenol and sodium azide suggesting that the process was energy independent. Furthermore, anionic exchangers may not be involved in the uptake process as DIDS, SITC and probenecid did not inhibit tyrosine uptake. Tyrosine uptake was observed to be saturable in micromolar range indicating the involvement of a saturable carrier system. Kinetic parameters, Km and Vmax were determined to be 34 ± 3 μM and 0.70 ± 0.02 nanomoles/min/mg protein, respectively. Similarly, saturation kinetics studies were also carried out in the presence of isoleucine, phenylalanine, leucine and alanine. Km and Vmax values obtained were 30 ± 2, 46 ± 3, 33 ± 4, 368 ± 12 μM and 4.10 ± 0.09, 3.53 ± 0.09, 3.37 ± 0.16, 2.10 ± 0.07 nanomoles/min/mg protein, respectively. Interestingly, no significant difference in the affinity (Km) values was observed for tyrosine, leucine, isoleucine and phenylalanine. However, the capacity (Vmax) differed significantly. LAT1 transporter capacity seemed to be maximal for isoleucine relative to tyrosine, leucine and phenylalanine.

BCH, a L system specific inhibitor, generated significant inhibition of L-tyrosine uptake in a concentration dependent manner. This inhibition data clearly indicate the involvement of system L for tyrosine uptake in PC-3 cells. Moreover, significant inhibition in tyrosine uptake was also observed in the presence of large neutral amino acids such as L-leucine, L-phenylalanine, L-tryptophan, L-isoleucine, L-methionine, L-histidine and L-valine. In addition, LAT1 specific substrates such as D-tyrosine, D-leucine, D-phenylalanine and D-tryptophan also produced significant inhibition of tyrosine uptake. These results clearly suggested the existence of LAT1 transporter for the tyrosine uptake.

In the present study, we also investigated the suitability of amino acid prodrug modification for enhancing drug accumulation in PC-3 cells. To delineate this process, we produced isoleucine-quinidine (Ile-quinidine) prodrug. We studied the intracellular accumulation of quinidine and Ile-quinidine in PC-3 cells. Ile-quinidine generated about two-fold increase in the uptake relative to quinidine.

In addition, we performed RT-PCR analysis to confirm the molecular expression of LAT1 and LAT2 in PC-3 cells. A 648 bp and 840 bp product band corresponding to the amplified human LAT1 and LAT2 was obtained in RT-PCR analysis (Fig. 1).

In conclusion, this study provides functional characterization and molecular expression of sodium independent LAT1 transporter in PC-3. Cellular accumulation studies demonstrated that Ile-quinidine generated a two-fold higher uptake relative to quinidine. Hence, amino acid modification approach might be a viable option to improve cellular accumulation of poorly permeable but highly potent anticancer drugs in prostate cancer cells.

Fig.-1Fig 1. RT-PCR analysis showing the expression of LAT1 and LAT2 in PC-3 cells. Lane 1: Molecular ladder. Lane 2: PCR product obtained for GAPDH as positive control. Lane 3, 4: PCR product at 840 and 648 bp obtained from total mRNA isolated from PC-3 cells with primers specific for human LAT2 (L2) and LAT1 (L1).

Multiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier SchönmannMultiselect Ultimate Query Plugin by InoPlugs Web Design Vienna | Webdesign Wien and Juwelier Schönmann