Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2

The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis–Menten constant (Km) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the Km values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a Ki value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H+ dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug–drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2.

► The methodology of transfected cells involved is simple, highly reproducible and results in low cytotoxicity.
► Transfected cells were used as an in vitro model to study the drug absorption/reabsorption mechanisms of PEPT1/PEPT2.
► HeLa-hPEPT1 and HeLa-hPEPT2 cells were used as an in vitro model to predict targets of the DDI between JBP485 and other substrates (such as cephalexin or lisinopril) of PEPT1 and PEPT2 in vivo.