The primary objective of this study is 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 assess the functional activity of LAT1. Reverse transcription-polymerase chain reaction (RT-PCR) analysis is carried out to confirm the molecular expression of LAT1. [3H]-tyrosine uptake is found to be time dependent and linear up to 60min. The uptake process does not exhibit any dependence on sodium ions, pH and energy. However, it is temperature dependent and found maximal at physiological temperature. The uptake of [3H]-tyrosine demonstrates saturable kinetics with K(m) and V(max) values of 34±3μM and 0.70±0.02nanomoles/min/mg protein, respectively. It is 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. Isoleucine-quinidine (Ile-quinidine) prodrug generates a significant inhibitory effect on [3H]-tyrosine uptake suggesting that it is recognized by LAT1. RT-PCR analysis provided a product band at 658 and 840bp, specific to LAT1 and LAT2, respectively. For the first time, this study demonstrates that LAT1, primarily responsible for the uptake of large neutral amino acids, is functionally active in PC-3 cells. Significant increase in the uptake generated by Ile-quinidine relative to quinidine suggests that LAT1 can be utilized for enhancing the cellular permeation of poor cell permeable anticancer drugs. Furthermore, this cell line can be utilized as an excellent in vitro model for studying the interaction of large neutral amino acid conjugated drugs with LAT1 transporter.
Written by:
Patel M, Dalvi P, Gokulgandhi M, Kesh S, Kohli T, Pal D, Mitra AK. Are you the author?
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108, USA.
Reference: Int J Pharm. 2013 Feb 25;443(1-2):245-53
doi: 10.1016/j.ijpharm.2012.12.029
PubMed Abstract
PMID: 23270998
