title: Functional characterization and molecular expression of large neutral amino acid transporter (LAT1) in human prostate cancer cells.
abstract: 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 60 min. 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 ± 3 μM and 0.70 ± 0.02 nanomoles/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 840 bp, 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.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, MO 64108, USA.
authors.forename: Mitesh
authors.initials: M
authors.lastname: Patel
authors.forename: Pranjali
authors.initials: P
authors.lastname: Dalvi
authors.forename: Mitan
authors.initials: M
authors.lastname: Gokulgandhi
authors.forename: Susamita
authors.initials: S
authors.lastname: Kesh
authors.forename: Tanvi
authors.initials: T
authors.lastname: Kohli
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D001692:Biological Transport; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics; D027283:Large Neutral Amino Acid-Transporter 1 / Q000096:biosynthesis / Q000502:physiology; D008297:Male; D011355:Prodrugs / Q000737:chemistry / Q000493:pharmacokinetics*; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D011802:Quinidine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000493:pharmacokinetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry; D013696:Temperature; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D027283:Large Neutral Amino Acid-Transporter 1; D011355:Prodrugs; C582850:isoleucine-quinidine conjugate; D007532:Isoleucine; D014443:Tyrosine; D011802:Quinidine