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Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2
Peptides 34 (2012) 395–403
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Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2 Xinjin Guo a , Qiang Meng a,c , Qi Liu a,c , Changyuan Wang a,c , Huijun Sun a,c , Taiichi Kaku b , Kexin Liu a,c,∗ a
Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, China Japan Bioproducts Industry Co. Ltd., Tomigaya, Shibuya-ku, Tokyo, Japan c Provincial Key Laboratory for Pharmacokinetics and Transport, Liaoning, Dalian Medical University, China b
a r t i c l e
i n f o
Article history: Received 16 January 2012 Received in revised form 10 February 2012 Accepted 10 February 2012 Available online 19 February 2012 Keywords: JBP485 Gly-Sar Transfected cells PEPT1 PEPT2 Uptake
a b s t r a c t 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. © 2012 Elsevier Inc. All rights reserved.
1. Introduction H+ -coupled peptide transporters, which are localized at the brush-border membranes of epithelial cells, mediate the absorption of various di- and tripeptides, as well as peptide-like drugs. As peptide transporters are primarily expressed in the small intestine and kidneys, the pharmacokinetic profiles of peptide-like drugs, i.e., their absorption rates and biological half-lives can be affected by the transport activities of peptide transporters. Two distinct peptide transporters, intestinal peptide transporter PEPT1 and renal PEPT2, have been cloned and characterized. Functional studies have confirmed that a wide variety of pharmacologically active peptide-like drugs, such as -lactam antibiotics and the angiotensin-converting enzyme inhibitors (ACEIs), are substrates
∗ Corresponding author at: 9 West Section, Lvshun South Road, Lvshunkou District, Dalian 116044, China. Tel.: +86 411 8611 0407; fax: +86 411 8611 0407. E-mail addresses: [email protected] (X. Guo), [email protected] (Q. Meng), [email protected] (Q. Liu), [email protected] (C. Wang), [email protected] (H. Sun), tai [email protected] (T. Kaku), [email protected] (K. Liu). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2012.02.009
of PEPT1 and PEPT2 [3,6,8,14,15,17]. Di- and tripeptides are taken up into intestinal cells by the low-affinity H+ -peptide cotransporter PEPT1. In the kidney tubule, di- and tripeptides are reabsorbed by PEPT1 and by the high-affinity H+ -peptide cotransporter PEPT2 [13,16]. Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide that was initially isolated from the human placenta hydrolyzate [9,10,20–23]. We previously reported that its absorption can be inhibited by glycylsarcosine (Gly-Sar), a dipeptide model drug and the substrate of Pepts [1,11,19]. This finding suggested that JBP485 was recognized by the peptide transporter system in the gastrointestinal tract and renal tubule. The purpose of the present study is to further confirm that JBP485 is the substrate of Pepts via the transfection of cells. To better screen, predict and understand the different routes of transport across the intestinal epithelium, several pharmaceutical cellular models have been reported to study oral absorption. The ability to isolate, clone and transfect the cDNAs for peptide transporters has provided a new dimension in developing cell culture models as screening tools. Studies conducted in either hPEPT1 adenovirally transfected Caco-2 cells or Chinese hamster
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ovary/hPEPT1 cells have demonstrated the utility of transfected cell lines for investigating the uptake of peptide transporter substrates [4,5]. In the present study, we constructed stably transfected HeLa-hPEPT1/HeLa-hPEPT2 clonal cell lines using the liposome transfection method. The methodology involved is simple, highly reproducible and results in low cytotoxicity. Furthermore, the transfected transporters will be used as an in vitro model to investigate the drug–drug interaction between JBP485 and other substrate (such as cephalexin) of PEPT1 and PEPT2 in our previous studies [22].
using this kit. Binding buffer and wash buffer were used to efficiently remove endotoxins from more than 0.1 mg of plasmid DNA. 2.4. Cell culture The parental HeLa cells were cultured in complete medium consisting of DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS (fetal bovine serum) without antibiotics, in an atmosphere of 5% CO2 and 95% air at 37 ◦ C. 2.5. Transfection
2. Materials and methods 2.1. Materials Gly-Sar and LipofectamineTM 2000 were purchased from Sigma–Aldrich (St. Louis, MO, USA). JBP485 was obtained from Japan Bioproducts Co. Ltd. (Toyko, Japan). HeLa cells were a gift from Dalian Medical University (Dalian, China). Endo-Free plasmid Midi Kit was purchased from Omega (USA). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM) and G418 were obtained from GIBCO Life Technologies (Grand Island, NY, USA). Ampicillin was purchased from Sigma–Aldrich. pBluescripthPEPT1 and pBluescript-hPEPT2 were kind donations from Yukio Kato (Kanazawa University, Kanazawa, Japan). HEPES, and all other chemicals, were obtained from Sigma–Aldrich. RIPA buffer was purchased from Beyotime Institute of Biotechnology (Haimen, China). BCA Kit (BCA Protein Assay Kit) was purchased from Solarbio (Beijing, China). Polyclonal antibody for hPEPT1/hPEPT2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence-plus reagents (from Beyotime Institute of Biotechnology, Haimen, China).
HeLa cells were seeded onto 24-Well plates. When the cells reached 70–80% confluence, the clonal HeLa-hPEPT1/HeLa-hPEPT2 cells were used as described previously. cDNAs encoding human PEPT1/PEPT2 were subcloned into the KpnI-, NotI and HindIII, and NotI-digested mammalian expression vector pcDNA3 and transfected into HeLa cells by the liposome transfection method according to the manufacturer’s protocol (LipofectamineTM 2000). G-418 (0.8 mg/ml)-resistant cells were picked, and PEPT1/PEPT2expressing cells (HeLa-hPEPT1/HeLa-hPEPT2) and mock cells (empty-plasmid transfected cells) were selected by measuring the Gly-Sar and JBP485 transport activities in 24-well plates 24 h posttransfection. The uptake medium was maintained at pH 6.0, as described previously [6]. 2.6. Western blot analysis
E. coli DH5␣ cells were grown in Luria–Bertani (LB) broth to early exponential phase and then pelleted by centrifugation at 1000 × g for 10 min at 4 ◦ C and resuspended in one-fifth of their original volume (in order to agitate) in ice-cold transformation and storage solution (TSS). A 0.1-ml aliquot of cells was then transferred into a cold polypropylene tube, mixed with 100 pg of pcDNA3.0 plasmid DNA, and incubated for 30 min at 40 ◦ C. Next, 0.9 ml of TSS, containing 20 mM of glucose, was added and the cells were grown at 37 ◦ C with shaking (225 rpm) for 1 h to allow expression of the antibiotic-resistance gene. Transformants were selected by plating cells (in triplicate) on agar plates containing ampicillin (30 mg/L). Transformation efficiencies (expressed as the number of transformants per g of DNA) were calculated after incubation of the plates at 37 ◦ C for 17–20 h. Transformants were screened for the presence of plasmid DNA by alkaline lysis of minipreps, restriction endonuclease digestion and gel electrophoresis. The input pcDNA3.0 DNA was recovered in all cases. No changes were detected in the restriction fragment sizes of the plasmid DNA, suggesting that TSS is not mutagenic for input DNA sequences. For the long-term storage of competent cells, cells suspended in TSS were frozen immediately in a dry ice/ethanol bath and stored at −70 ◦ C until needed. Frozen cells were thawed on ice and used immediately in the transformation assay. TSS consisted of PEG (polyethylene glycols, molecular weight: 3350), 5% (v/v) DMSO (dimethyl sulfoxide), and 40 mM Mg2+ (MgCl2 ), at a final pH of 6.5.
For Western blot analysis, the incubation medium of HeLahPEPT1/HeLa-hPEPT2 cells was removed and the cells were rapidly rinsed three times with 3 ml of ice-cold PBS (phosphate-buffered saline). 24-well plates that had been frozen by the ice pack and stored in RIPA (rapid immunoprecipitation assay) buffer containing protease inhibitors (proteinase inhibitors and phosphatase inhibitors). The lysate was centrifuged at 12,000 × g for 30 min to remove cell debris and collect the supernatant. Protein was measured according to the procedure of BCA (bicinchoninic acid) Kit with bovine serum albumin as the standard. Equal amounts of 10 g of proteins were resuspended in electrophoresis sample buffer containing -mercaptoethanol and separated by electrophoresis on a pre-cast 10% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA), followed by electrotransfer to a PVDF membrane (Millipore, Bedford, MA). Membranes were blocked using 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST). -Actin served as loading control. Membranes were incubated overnight at 4 ◦ C with a 1:500 dilution of polyclonal antibody for hPEPT1/hPEPT2, and with a 1:2000 dilution of monoclonal antibody for -actin (Santa Cruz Biotechnology, Santa Cruz, CA). After washing three times in TBST, membranes were incubated for 1 h at 37 ◦ C with a 1:5000 dilution of anti-rabbit or a 1:1000 dilution of anti-mouse horseradish peroxidase-conjugated secondary antibody respectively (Invitrogen, Shanghai, China). After extensive washing with TBST, membranes were exposed to the enhanced chemiluminescenceplus reagents (ECL) according to the manufacturer’s protocol. Emitted light was documented with a BioSpectrum-410 multispectral imaging system with a Chemi HR camera 410. Protein bands were visualized and photographed under transmitted ultraviolet light. The image was used for semiquantitative measurements based on band densitometry. RIPA buffer consisted of 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS (sodium dodecyl sulfate).
2.3. Extraction and purification of plasmid DNA
2.7. Uptake studies using cell monolayers
Endo-Free plasmid Midi Kit was used to isolate plasmid DNA from bacterial cultures. Up to 200 ml of culture can be processed
The uptake of Gly-Sar and JBP485 was measured in cells grown on 24-well plates, as described previously. The composition of the
2.2. Transformation procedure
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incubation medium was as follows (in mM): 145 NaCl, 3.0 KCl, 1.0 CaCl2 , 0.5 MgCl2 and 5 d-glucose. The pH of the medium was adjusted with a solution of NaOH or HCl. The cells were preincubated for 15 min at 37 ◦ C with 1 ml of the incubation medium. After removal of the medium, the cells were incubated with 1 ml of incubation medium (pH 6.0) containing Gly-Sar or JBP485 (0.5 mM for PEPT1, 0.1 mM for PEPT2) for 3 min at 37 ◦ C. After incubation, the medium was aspirated, and the cells were rapidly rinsed three times with 3 ml of ice-cold PBS (pH 7.4) and lysed in 0.3 ml/well of PBS with 1% Triton X-100 for 60 min. The aliquots were subjected to both LC–MS/MS (liquid chromatography–mass spectrometry), as described below, and protein quantification using the BCA Kit. Uptake activity was determined as the concentration of Gly-Sar and JBP485 by LC–MS/MS per ml of protein every 3 min. To measure the Gly-Sar transport kinetics, Gly-Sar or JBP485 uptake, within a concentration range of 0.050–10 mM and 0.010–5 mM respectively, was assessed with an incubation time of 3 min. 2.8. Drug–drug interaction using cell monolayers HeLa-hPEPT1/HeLa-hPEPT2 cells were grown in DMEM with 10% FBS with antibiotics in an atmosphere of 5% CO2 /95% air at 37 ◦ C, respectively. The assays were carried out in 24-well culture plates, with nearly confluent cells seeded 48 h before each experiment. Immediately before the experiment, the cells were washed twice with 1 ml HBSS (Hank’s balanced salt solution) buffer at room temperature and then incubated with 1 ml HBSS for 15 min at 37 ◦ C. The pH of the medium was adjusted with a solution of NaOH or HCl. After removal of the medium, the cells were incubated with 1 ml HBSS (pH 5.8 for HeLa-hPEPT1 cells and pH 6.5 for HeLa-hPEPT2 cells) containing drugs including: (1) JBP485 alone (0.5 mM for HeLa-hPEPT1 cells) as control of JBP 485; (2) cephalexin alone (1 mM for HeLa-hPEPT1 cells) as control of cephalexin; (3) JBP485 (0.5 mM) + cephalexin (1 mM) for HeLa-hPEPT1 cells as the inhibitory group; (4) JBP485 alone (0.1 mM for HeLa-hPEPT2 cells) as control of JBP485; (5) lisinopril alone (0.02 mM for HeLa-hPEPT2 cells) as control of lisinopril; or (6) JBP485 (0.1 mM) + lisinopril (0.02 mM for HeLa-hPEPT2 cells) as the inhibitory group. After incubation for the designated times at 37 ◦ C with gentle shaking, the experiment was terminated by removing the medium, followed by washing three times with 1 ml ice-cold HBSS and lysed in 0.3 ml/well HBSS with 1% Triton X-100 for 60 min. The aliquots were subjected to LC–MS/MS. The uptake assays of the two agents were measured in a time-dependent manner, at the uptake time of 1, 3, 5, 10 and 15 min for HeLa-hPEPT1 cells and HeLa-hPEPT1 cells. HBSS consisted of 8 g/L NaCl, 0.4 g/L KCl, 1 g/L d-glucose, 60 mg/L KH2 PO4 , 47.5 mg/L Na2 HPO4 and 1000 ml tri-distilled water. The pH = 7.2 of the medium was adjusted with a solution of NaOH or HCl. 2.9. Biological sample preparation The concentrations of JBP485, Gly-Sar, cephalexin (CEX) and lisinopril in cell lysates were determined by LC–MS/MS. A 50-l aliquot of internal standard (IS, 500 ng/ml, JBP923 for JBP485, GlySar and cephalexin; paracetamol for lisinopril) and 500 l methanol were added to 25 l cell lysates. The mixture was vortexed for 1 min and centrifuged at 16,099 × g for 10 min to remove the protein precipitate; the upper organic layer was transferred into a polythene tube and dried with nitrogen at 37 ◦ C. The dried residue was dissolved by mobile phase, and 10 l was injected into the LC–MS/MS. 2.10. LC–MS/MS analysis An Agilent LC systems (Agilent HP1200, Agilent Technology Inc., Palo Alto, CA, USA) was used. Isocratic chromatographic separation
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was performed on a Hypersil BDS-C18 column 150 mm × 4.6 mm i.d., 5 m (Dalian Elite Analytical Instruments Co. Ltd., China). The mobile phase consisted of 3% methanol and 97% water with 0.1% formic acid for JBP 485, Gly-Sar, cephalexin and JBP923 (IS), 30% methanol and 70% water with 0.1% formic acid for lisinopril and paracetamol (IS). The flow rate was 0.5 ml/min. The column was maintained at room temperature. An API 3200 triple-quadrupole mass spectrometer (Applied Biosystems, Concord, Ont, Canada) was operated with a TurboIonspray interface in positive ion mode. Analyst 1.4.1 software (Applied Biosystems) was used for the control of equipment, data acquisition and analysis. For the optimization of MS/MS parameters, the instrument was operated with an ion spray voltage at +4.5 kV, heater gas temperature at 450 ◦ C, nebulizer gas (Gas 1) at 0.34 MPa, heater gas (Gas 2) at 0.34 MPa, curtain gas at 0.060 MPa and collision gas at 0.030 MPa. All gases used were nitrogen. Declustering potential was set at 35 V for both the analytes and IS. Multiple reaction monitoring (MRM) was employed for data acquisition. The optimized ‘truncated’ MRM fragmentation transitions were m/z 201.1 → m/z 86.1 with collision energy (CE) of 33 eV for JBP 485, m/z 147.1 → m/z 60.1 with CE of 40 eV for Gly-Sar, m/z 348.1 → m/z 174.1 with CE of 22 eV for CEX, m/z 219.1 → m/z 86.1 with CE of 38 eV for JBP923 (IS), m/z 406.1 → m/z 206.0 with CE of 30 eV for lisinopril and m/z 152.1 → m/z 110.1 with CE of 24 eV for paracetamol (IS). The dwell time for each transition was 200 ms. 2.11. Protein assay After solubilizing cells in 1% Triton X-100, 10 l of cell lysates was taken from each well for protein concentration determination using the BCA protein assay kit (Solarbio), according to the manufacturer’s instructions. Bovine serum albumin (BSA) was used as a standard. 2.12. Statistical analysis All data are given as the mean ± SD of three independent experiments. The kinetic parameters were calculated using nonlinear regression methods and confirmed by linear regression of the respective Eadie–Hofstee plots. IC50 (i.e., the concentration of the compound necessary to inhibit 50% of specific substrate carriermediated uptake) values were determined by nonlinear regression using the logistical equation for an asymmetric sigmoid (allosteric Hill kinetics): Y = Min + (Max − Min)/(1 + (X/IC50 )P ), where Max is the initial Y value, Min the final Y value, and the power P represents the Hill coefficient. Inhibition constants (Ki ) were calculated from IC50 values according to the method developed by Cheng and Prusoff: IC50 = Ki (1 + [S]/Km ) [2,7]. Statistical analysis was carried out using the SPSS11.5 package. Test results were expressed as mean ± SD. To test for statistically significant differences among multiple treatments for a given parameter, one-way analysis of variance (ANOVA) was performed. The statistical significance of differences between mean values was calculated using the non-paired t-test. If p was <0.05 or <0.01, differences were considered statistically significant. 3. Results 3.1. Transformation, extraction and purification of plasmid After transformation, plasmid was prepared from 200 ml of bacterial suspension. After extraction and purification steps, a large amount of pure plasmid DNA was obtained. The approximate concentrations of the plasmids were 0.8 mg/ml for pcDNA 3.0-hPEPT1 and 0.4 mg/ml for pcDNA 3.0-hPEPT2, as determined by agarose gel electrophoresis (Fig. 1). The pcDNA 3.0 expression vector with
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Fig. 1. Identification and semiquantitative analysis of pcDNA3-hPEPT1/-hPEPT2 by agarose gel electrophoresis. (A) pcDNA3/PEPT1 (+); (B) pcDNA3/PEPT1 (−); (C) pcDNA3/PEPT2 (+); (D) pcDNA3/PEPT2 (−).
the human PEPT1/PEPT2 cDNA inserted was transfected into HeLa cells, and approximately 0.8 mg/L of G418-resistant cells were selected and examined for Gly-Sar or JBP485 transport activities. Clones with the highest transport activities was selected (HeLahPEPT1/HeLa-hPEPT2 cells) and used for further characterization of human PEPT1/PEPT2.
3.2. Identification of HeLa-hPEPT1 and HeLa-hPEPT2 cells by Western blot analysis To confirm that the stably transfected HeLa-hPEPT1/HeLahPEPT2 cells (human PEPT1/PEPT2-expressing HeLa cells) were constructed successfully, the protein expression levels of hPEPT1 and HPEPT2 were examined by Western blot analysis. As shown in Fig. 2, PEPT1 and PEPT2 were found almost exclusive in HeLahPEPT1 and HeLa-hPEPT2 cells, respectively, but not found in the mock cell.
Fig. 2. Identification analysis of hPEPT1/hPEPT2 transfected cells by Western blot. (A) HeLa-hPEPT1 cells and (B) HeLa-hPEPT2 cells.
3.3. Characteristics of Gly-Sar or JBP485 uptake by HeLa-hPEPT1 and HeLa-hPEPT2 cells Time dependence assay. First, we examined the time course of Gly-Sar or JBP485 uptake at pH 6.0 by HeLa-hPEPT1 (human PEPT1expressing HeLa cells) and HeLa-hPEPT2 cells. The rate of Gly-Sar or JBP485 uptake in HeLa-hPEPT1 cells was much greater than that observed in HeLa-hPEPT2 cells (Fig. 3). On the other hand, the uptake of Gly-Sar or JBP485 by the parental HeLa cells transfected with control vector (HeLa-empty vector) was negligible compared with that by HeLa-hPEPT1 and HeLa-hPEPT2 cells. Linear uptakes were detected up to 3 min for HeLa-hPEPT1 cells and HeLa-hPEPT2 cells, respectively. pH dependence assay. To confirm that uptake of JBP485 and Gly-Sar by HeLa-hPEPT1 and HeLa-hPEPT2 cells was dependent on hydrogen ions, the effects of pH of the medium on Gly-Sar or JBP485 uptake by HeLa-hPEPT1 and HeLa-hPEPT2 cells were examined. In HeLa-hPEPT1 cells, Gly-Sar or JBP485 uptake was maximal at pH 5.8, whereas it was maximal at pH 6.5 in HeLa-hPEPT2 cells
Fig. 3. Time dependence of Gly-Sar or JBP485 uptake by hPEPT1-HeLa cell (A and C) and hPEPT2-HeLa cell (B and D). Cells were incubated at 37 ◦ C with Gly-Sar (A and B) or JBP485 (C and D) (A and C, 0.5 mM; B and D, 0.1 mM). Each symbol and bar represents the mean ± SD of three experiments.
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Fig. 4. pH dependence of Gly-Sar uptake by hPEPT1-HeLa cell (A) and hPEPT2-HeLa cell (B). Cells were incubated at 37 ◦ C with Gly-Sar (A, 0.5 mM; B, 0.1 mM). Each symbol and bar represents the mean ± SD of three experiments.
(Fig. 4). These data suggested uptake of JBP485 and Gly-Sar by the H+ /peptide symporter PEPT1 and PEPT2. We determined the uptake of Gly-Sar or JBP485 at pH 5.8 for HeLa-hPEPT1 cells and at pH 6.5 for HeLa-hPEPT2 cells in our subsequent experiments. Concentration dependence assay. To further confirm that our transfected models had been constructed successfully, the concentration dependence of Gly-Sar or JBP485 uptake in HeLahPEPT1/HeLa-hPEPT2 cells was determined. Specific uptake was calculated by subtracting the nonspecific uptake, which was estimated in the presence of excess unlabeled dipeptide, from the total uptake, and kinetic parameters were calculated according to the Michaelis–Menten equation. The Michaelis–Menten constant (Km ) values of Gly-Sar uptake by the hPEPT1 and hPEPT2expressing transfectants were 1.03 mM and 0.0965 mM (Fig. 5A and B, insets), respectively, and the Km values of JBP485 uptake were 1.33 mM (for PEPT1) and 0.144 mM (for PEPT2) (Fig. 5C and D, insets). These results suggested that human PEPT2 has 10-fold higher affinity for Gly-Sar or JBP485 uptake than PEPT1. The maximum velocity (Vmax ) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.86 nmol/mg protein/3 min and 0.664 nmol/mg protein/3 min, respectively, and the Vmax values of JBP485 uptake were 1.75 nmol/mg protein/3 min and 0.545 nmol/mg protein/3 min, respectively. 3.4. Mutual inhibition between Gly-Sar and JBP485 in HeLa-hPEPT1/HeLa-hPEPT2 cells To determine the targets of the interaction between Gly-Sar and JBP485, the concentration-dependent inhibition of Gly-Sar uptake
by JBP485 in the HeLa-hPEPT1 (Fig. 6A) and HeLa-hPEPT2 (Fig. 6B) cells, as well as the concentration-dependent inhibition of JBP485 uptake by Gly-Sar in the HeLa-hPEPT1 (Fig. 6C) and HeLa-hPEPT2 (Fig. 6D) cells, were investigated. The uptake of Gly-Sar was significantly inhibited by JBP485 with a Ki (inhibition constants) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). The uptake of JBP485 was also significantly inhibited by Gly-Sar with a Ki value of 12.9 mM (for PEPT1) and 2.98 mM (for PEPT2). These findings suggested that the targets of interaction between Gly-Sar and JBP485 were PEPT1 and PEPT2. 3.5. Drug–drug interaction between JBP485 and cephalexin in HeLa-hPEPT1 cells To investigate that the target of DDI between JBP485 and cephalexin was related to PEPT1 in the intestine, HeLa-hPEPT1 cells were used to examine the uptake of JBP 485 and cephalexin in the hPEPT1-transfected cells. The uptake concentration of JBP485 or cephalexin in HeLa-hPEPT1 cells was significantly reduced (Fig. 7A and B) when the two drugs were co-administered. On the other hand, the uptake of JBP485 or cephalexin by the mock cells was negligible compared with the uptake by HeLa-hPEPT1 cells. 3.6. Drug–drug interaction between JBP485 and lisinopril in HeLa-hPEPT2 cells To investigate that the target of DDI between JBP485 and lisinopril is PEPT2 in the kidneys, we investigated the uptake interaction of JBP485 and lisinopril in HeLa-hPEPT2 cells. Lisinopril markedly
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Fig. 5. Concentration dependence of Gly-Sar or JBP485 uptake by hPEPT1-HeLa cell (A and C) and hPEPT2-HeLa cell (B and D). Cells were incubated for 3 min at 37 ◦ C with Gly-Sar (A and B) or JBP485 (C and D). Each symbol and bar represents the mean ± SD of three experiments. Insets: Eadie–Hofstee plots of the uptake after correction for the nonsaturable component; V, Gly-Sar/JBP485 uptake rate (nmol/mg protein/3 min); S, Gly-Sar/JBP485 concentration (mM).
inhibited the uptake of JBP485 in HeLa-hPEPT2 cells (Fig. 8A). On the other hand, JBP485 also markedly inhibited the uptake of lisinopril in HeLa-hPEPT2 cells (Fig. 8B). The uptake of JBP485 or lisinopril was significantly greater in HeLa-hPEPT2 cells than in the mock cells.
4. Discussion Peptide transporters PEPT1 and PEPT2 have been demonstrated to play important physiological and nutritional roles in cells, and have also been shown to be of pharmacokinetic and pharmacological significance. PEPT1/PEPT2 not only serves to mediate the absorption/reabsorption of nutrients but also functions in the transport of exogenous compounds that have peptide-like structures, such as ACEIs and some -lactam antibiotics [6,12]. Therefore, when substrates of PEPT1/PEPT2 combine, their pharmacological effects may be altered. In the present study, to investigate targets in the absorption/reabsorption of these substrates, we established HeLa cells stably transfected with human PEPT1/PEPT2 cDNA and therefore expressing a proposed protoncoupled dipeptide transporter. Transporter-expressing cell models have proved to be high throughput approaches that can rapidly provide the information for identifying interaction between a compound and a particular transporter. Several attempts to develop a cell culture model having enhanced peptide transport activity have been taken by
use of cloning and recombinant DNA technology and some overexpression systems have well established. For instance, hPEPT1 has been functionally expressed in Caco-2 cells and also stably transfected into the CHO cells [4,5]. In our work, the human PEPTs transporter expressed in HeLa cells was developed. Compared to CHO cells, HeLa cells are human-derived cells which can avoid species differences. Compared to Caco-2 cells, HeLa cells have a low expression level of PEPTs, eliminating the possibility that an endogenous transporter could complicate interpretation of experiments. In our study, mock cells (empty-vector transfected HeLa cells) have little baseline uptake of Gly-Sar and JBP485 which have been improved to be a substrate of hPEPT1 and hPEPT2 in our previous experiment. There are various methods for the introduction of DNA or RNA molecules into cultured mammalian cells. These methods include chemical (liposome-mediated), physical (electroporation, microinjection, heat shock) or viral-based (retrovirus, adeno-associated virus) delivery systems. The liposome-mediated transfection method has several advantages. First, the method is simple and highly reproducible. Second, exogenous DNAs can be introduced into large numbers of cells, and third, the liposomes exhibit low cytotoxicity. To confirm that the transfected cells had been constructed successfully, we performed assays to investigate the protein expression of PEPT1/PEPT2 and the time, pH and concentration dependence of Gly-Sar and JBP485 uptake. First, by Western blot analysis of protein expression, PEPT1 and PEPT2 were highly
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Fig. 6. Interaction between Gly-Sar and JBP485 in hPEPT1-HeLa cell (A and C) and hPEPT2-HeLa cell (B and D). The uptake of Gly-Sar (A, 0.5 mM; B, 0.1 mM) was inhibited by JBP485 (A, 0.5–50 mM; B, 0.1–10 mM). The uptake of JBP485 (C, 0.5 mM; D, 0.1 mM) was inhibited by Gly-Sar (C, 0.5–50 mM; D, 0.1–10 mM). Each symbol and bar represents the mean ± SD of three experiments.
expressed in HeLa-hPEPT1 cells and HeLa-hPEPT2 cells relative to the mock cells, respectively, which confirmed that the transfected cell were constructed successfully on the protein level (Fig. 2). hPEPT1-HeLa cells were selected by measuring the uptake activities of Gly-Sar and JBP485. As shown in Fig. 3, significantly greater accumulation of Gly-Sar or JBP485 was detected in hPEPT1-HeLa
cells than in the empty vector-containing cells. Linear uptakes were recorded before 3 min. Similar results were obtained in the time dependence assays for uptake of Gly-Sar or JBP485 in hPEPT2-HeLa cells (Fig. 3B and D). Differences in the substrate affinity and pH dependence of PEPT1 and PEPT2 for Gly-Sar and JBP485 uptake suggested that these transporters play distinct roles in intestinal
Fig. 7. Time profiles of the uptake of JBP485 (A) and cephalexin (B, CEX) by HeLa–hPEPT1 cells. The time-dependent uptake of JBP485 (0.5 mM) and cephalexin (CEX, 1 mM) by HeLa–hPEPT1 cells were examined at 37 ◦ C. (Each symbol and bar represents the mean ± SD of three experiments; *p < 0.05 vs. control, **p < 0.01 vs. control.)
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Fig. 8. Time profiles of the uptake of JBP485 (A) and lisinopril (B) by HeLa–hPEPT2 cells. The time-dependent uptake of JBP485 (0.1 mM) and lisinopril (0.02 mM) by HeLa–hPEPT2 cells were examined at 37 ◦ C. (Each symbol and bar represents the mean ± SD of three experiments; *p < 0.05 vs. control, **p < 0.01 vs. control.)
absorption and tubular reabsorption of oligopeptides. The uptake of Gly-Sar or JBP485 in HeLa-hPEPT1 cells was maximal at pH 5.8, whereas it was maximal at pH 6.5 in HeLa-hPEPT2 cells. These results suggested that HeLa-hPEPT1 cells prefer a more acidic pH to transport Gly-Sar or JBP485 than HeLa-hPEPT2 cells (Fig. 4). Furthermore, the results confirmed that both hPEPT1 and hPEPT2 were proton-coupled dependence transporters. The Michaelis–Menten Km values of PEPT1 and PEPT2 for Gly-Sar uptake were similar to previously reported values (Km value of 1.1 mM in LLC-rPEPT1 cells and 0.11 mM in LLC-rPEPT2 cells for Gly-Sar) [18]. By contrast, the Km values for JBP485 uptake were found to be 1.33 mM for PEPT1 and 0.144 mM for PEPT2 in the human PEPT1- and PEPT2expressing transfectants. These findings confirm that PEPT2 is the high-affinity type transporter in the kidney, whereas PEPT1 is the low-affinity type transporter in the small intestine. Importantly, our findings revealed that human PEPT1 and PEPT2 have different characteristics, not only regarding substrate affinity for native dipeptide, but also in the recognition of a variety of peptide-like drugs. As mentioned above, the Km values for Gly-Sar uptake by HeLahPEPT1/hPEPT2 cells were significantly lower than those for JBP485 uptake. Gly-Sar is a l-l-dipeptides and has a peptide bond with trans conformation. PEPT1 is stereospecific in the sense that ll-dipeptides and l-l-l-tripeptides display a much higher affinity than dipeptides or tripeptides containing d-amino acids [24]. The structure of Gly-Sar was a peptide bond with trans conformation. A previous study investigated the influence of the substrate backbone dynamics caused by peptide bond cis/trans isomerization on the intestinal peptide transport [25]. Trans contents and affinities for PEPT1 were positively correlated. It was concluded that PEPT1 accepts trans conformers of zwitterionic dipeptides and derivatives thereof regardless of size, hydrophobicity and aromatic nature of the N-terminal amino acid. Therefore, the interaction between GlySar and the peptide transporter would be much stronger than the interaction between cyclo-dipeptide and the peptide transporter. Clinical drug–drug interactions are expected to enhance the beneficial effects of a particular drug or reduce its toxicity. However, some drug combinations will enhance the toxic effects, or reduce the pharmacologic effects; these are undesirable drug–drug interactions. 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. When JBP485 or cephalexin was used alone, the uptake concentration of JBP485 or cephalexin was significantly higher than that in the co-administered groups. This result
indicated that the target of DDI between JBP485 and cephalexin was related, at least in part, to PEPT1. The uptake of JBP485 or lisinopril was inhibited significantly when the two drugs were added simultaneously to the HeLa-hPEPT2 cells. This result indicated that the targets of DDI between JBP485 and lisinopril were, at least in part, PEPT2 in the kidneys. Our findings provide useful information regarding potential drug design strategies and delivery systems, which may be used to improve the efficiency of drug therapy. Our results may also prove important in appropriately interpreting and predicting the potential for transporter-associated DDI and toxicity. 5. Conclusion (1) Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully. (2) The functions of the transfected cells were identified using the substrates JBP485 and Gly-Sar. (3) 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. Acknowledgements This work was supported in part by a grant from the National Natural Science Foundation of China (No. 81072694) and Dalian Government (Nos. 2009E12SF155 and 2010E12SF060). We wish to express our deep gratitude to Yukio Kato (Kanazawa University, Kanazawa, Japan) for providing pcDNA3-PEPT1 and pcDNA3PEPT2. References [1] Cang J, Zhang J, Wang CY, Liu Q, Meng Q, Wand D, et al. Pharmacokinetics and mechanism of intestinal absorption of JBP485 in rats. Drug Metab Pharmacokinet 2010;25:500–7. [2] Cheng Y, Prusoff WH. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 1973;22:3099–108. [3] Daniel H, Adibi SA. Transport of -lactam antibiotics in kidney brush border membrane. Determinants of their affinity for the oligopeptide/H1 symporter. J Clin Invest 1993;92:2215–23. [4] Han HK, Rhie JK, Oh DM, Saito G, Hsu CP, Stewart BH, et al. CHO/hPEPT1 cells overexpressing the human peptide transporter (hPEPT1) as an alternative in vitro model for peptidomimetic drugs. J Pharm Sci 1999;88:347–50. [5] Hsu CP, Walter E, Merkle HP, Rothen-Rutishauser B, Wunderli-Allenspach H, Hilfinger JM, et al. Function and immunolocalization of overexpressed human
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[16] Terada T, Inui KI. Peptide transporters: structure, function, regulation and application for drug delivery. Curr Drug Metab 2004;5:85–94. [17] Terada T, Saito H, Mukai M, Inui K. Recognition of -lactam antibiotics by rat peptide transporters, PEPT1 and PEPT2, in LLC-PK1 cells. J Pharmacol Exp Ther 1997;281:1415–21. [18] Terada T, Saito H, Mukai M, Inui KI. Recognition of -lactam antibiotics by rat peptide transporters, PEPT1 and PEPT2, in LLC-PK1 cells. Am J Physiol Renal Physiol 1997;273:F706–11. [19] Wang W, Liu Q, Wang CY, Meng Q, Kaku T, Liu KX. Effect of JBP485 on the expression and function of PEPT1 in indomethacin-induced intestinal injury in rats and damage in Caco-2 cells. Peptides 2011;32:946–55. [20] Wu JJ, Wang CY, Liu Q, Yang T, Zhang QH, Peng JY, et al. Protective effect of JBP485 on concanavalin A-induced hepatocyte toxicity in primary cultured rat hepatocytes. Eur J Pharmacol 2008;589:299–305. [21] Yang T, Wu JJ, Wang CY, Liu Q, Ma XC, Peng JY, et al. Protective effect of JBP485 on concanavalin A-induced liver injury in mice. J Pharm Pharmacol 2009;61:767–74. [22] Zhang J, Wang CY, Liu Q, Meng Q, Cang J, Sun HJ, et al. Pharmacokinetic interaction between JBP485 and cephalexin in rats. Drug Metab Dispos 2010;38:930–8. [23] Zhang Q, Liu Q, Wu J, Wang C, Peng J, Ma X, et al. PEPT1 involved in the uptake and transepithelial transport of cefditoren in vivo and in vitro. Eur J Pharmacol 2009;612:9–14. [24] Lister N, Sykes AP, Bailey PD, Boyd CA, Bronk JR. Dipeptide transport and hydrolysis in isolated loops of rat small intestine: effects of stereospecificity. J Physiol 1995;1:173–82. [25] Brandsch M, Knütter I, Leibach FH. The intestinal H+ /peptide symporter PEPT1: structure–affinity relationships. Eur J Pharm Sci 2004;21:53–60.
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Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2 Xinjin Guo a , Qiang Meng a,c , Qi Liu a,c , Changyuan Wang a,c , Huijun Sun a,c , Taiichi Kaku b , Kexin Liu a,c,∗ a
Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, China Japan Bioproducts Industry Co. Ltd., Tomigaya, Shibuya-ku, Tokyo, Japan c Provincial Key Laboratory for Pharmacokinetics and Transport, Liaoning, Dalian Medical University, China b
a r t i c l e
i n f o
Article history: Received 16 January 2012 Received in revised form 10 February 2012 Accepted 10 February 2012 Available online 19 February 2012 Keywords: JBP485 Gly-Sar Transfected cells PEPT1 PEPT2 Uptake
a b s t r a c t 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. © 2012 Elsevier Inc. All rights reserved.
1. Introduction H+ -coupled peptide transporters, which are localized at the brush-border membranes of epithelial cells, mediate the absorption of various di- and tripeptides, as well as peptide-like drugs. As peptide transporters are primarily expressed in the small intestine and kidneys, the pharmacokinetic profiles of peptide-like drugs, i.e., their absorption rates and biological half-lives can be affected by the transport activities of peptide transporters. Two distinct peptide transporters, intestinal peptide transporter PEPT1 and renal PEPT2, have been cloned and characterized. Functional studies have confirmed that a wide variety of pharmacologically active peptide-like drugs, such as -lactam antibiotics and the angiotensin-converting enzyme inhibitors (ACEIs), are substrates
∗ Corresponding author at: 9 West Section, Lvshun South Road, Lvshunkou District, Dalian 116044, China. Tel.: +86 411 8611 0407; fax: +86 411 8611 0407. E-mail addresses: [email protected] (X. Guo), [email protected] (Q. Meng), [email protected] (Q. Liu), [email protected] (C. Wang), [email protected] (H. Sun), tai [email protected] (T. Kaku), [email protected] (K. Liu). 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2012.02.009
of PEPT1 and PEPT2 [3,6,8,14,15,17]. Di- and tripeptides are taken up into intestinal cells by the low-affinity H+ -peptide cotransporter PEPT1. In the kidney tubule, di- and tripeptides are reabsorbed by PEPT1 and by the high-affinity H+ -peptide cotransporter PEPT2 [13,16]. Cyclo-trans-4-l-hydroxyprolyl-l-serine (JBP485) is a dipeptide that was initially isolated from the human placenta hydrolyzate [9,10,20–23]. We previously reported that its absorption can be inhibited by glycylsarcosine (Gly-Sar), a dipeptide model drug and the substrate of Pepts [1,11,19]. This finding suggested that JBP485 was recognized by the peptide transporter system in the gastrointestinal tract and renal tubule. The purpose of the present study is to further confirm that JBP485 is the substrate of Pepts via the transfection of cells. To better screen, predict and understand the different routes of transport across the intestinal epithelium, several pharmaceutical cellular models have been reported to study oral absorption. The ability to isolate, clone and transfect the cDNAs for peptide transporters has provided a new dimension in developing cell culture models as screening tools. Studies conducted in either hPEPT1 adenovirally transfected Caco-2 cells or Chinese hamster
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ovary/hPEPT1 cells have demonstrated the utility of transfected cell lines for investigating the uptake of peptide transporter substrates [4,5]. In the present study, we constructed stably transfected HeLa-hPEPT1/HeLa-hPEPT2 clonal cell lines using the liposome transfection method. The methodology involved is simple, highly reproducible and results in low cytotoxicity. Furthermore, the transfected transporters will be used as an in vitro model to investigate the drug–drug interaction between JBP485 and other substrate (such as cephalexin) of PEPT1 and PEPT2 in our previous studies [22].
using this kit. Binding buffer and wash buffer were used to efficiently remove endotoxins from more than 0.1 mg of plasmid DNA. 2.4. Cell culture The parental HeLa cells were cultured in complete medium consisting of DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FBS (fetal bovine serum) without antibiotics, in an atmosphere of 5% CO2 and 95% air at 37 ◦ C. 2.5. Transfection
2. Materials and methods 2.1. Materials Gly-Sar and LipofectamineTM 2000 were purchased from Sigma–Aldrich (St. Louis, MO, USA). JBP485 was obtained from Japan Bioproducts Co. Ltd. (Toyko, Japan). HeLa cells were a gift from Dalian Medical University (Dalian, China). Endo-Free plasmid Midi Kit was purchased from Omega (USA). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM) and G418 were obtained from GIBCO Life Technologies (Grand Island, NY, USA). Ampicillin was purchased from Sigma–Aldrich. pBluescripthPEPT1 and pBluescript-hPEPT2 were kind donations from Yukio Kato (Kanazawa University, Kanazawa, Japan). HEPES, and all other chemicals, were obtained from Sigma–Aldrich. RIPA buffer was purchased from Beyotime Institute of Biotechnology (Haimen, China). BCA Kit (BCA Protein Assay Kit) was purchased from Solarbio (Beijing, China). Polyclonal antibody for hPEPT1/hPEPT2 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Enhanced chemiluminescence-plus reagents (from Beyotime Institute of Biotechnology, Haimen, China).
HeLa cells were seeded onto 24-Well plates. When the cells reached 70–80% confluence, the clonal HeLa-hPEPT1/HeLa-hPEPT2 cells were used as described previously. cDNAs encoding human PEPT1/PEPT2 were subcloned into the KpnI-, NotI and HindIII, and NotI-digested mammalian expression vector pcDNA3 and transfected into HeLa cells by the liposome transfection method according to the manufacturer’s protocol (LipofectamineTM 2000). G-418 (0.8 mg/ml)-resistant cells were picked, and PEPT1/PEPT2expressing cells (HeLa-hPEPT1/HeLa-hPEPT2) and mock cells (empty-plasmid transfected cells) were selected by measuring the Gly-Sar and JBP485 transport activities in 24-well plates 24 h posttransfection. The uptake medium was maintained at pH 6.0, as described previously [6]. 2.6. Western blot analysis
E. coli DH5␣ cells were grown in Luria–Bertani (LB) broth to early exponential phase and then pelleted by centrifugation at 1000 × g for 10 min at 4 ◦ C and resuspended in one-fifth of their original volume (in order to agitate) in ice-cold transformation and storage solution (TSS). A 0.1-ml aliquot of cells was then transferred into a cold polypropylene tube, mixed with 100 pg of pcDNA3.0 plasmid DNA, and incubated for 30 min at 40 ◦ C. Next, 0.9 ml of TSS, containing 20 mM of glucose, was added and the cells were grown at 37 ◦ C with shaking (225 rpm) for 1 h to allow expression of the antibiotic-resistance gene. Transformants were selected by plating cells (in triplicate) on agar plates containing ampicillin (30 mg/L). Transformation efficiencies (expressed as the number of transformants per g of DNA) were calculated after incubation of the plates at 37 ◦ C for 17–20 h. Transformants were screened for the presence of plasmid DNA by alkaline lysis of minipreps, restriction endonuclease digestion and gel electrophoresis. The input pcDNA3.0 DNA was recovered in all cases. No changes were detected in the restriction fragment sizes of the plasmid DNA, suggesting that TSS is not mutagenic for input DNA sequences. For the long-term storage of competent cells, cells suspended in TSS were frozen immediately in a dry ice/ethanol bath and stored at −70 ◦ C until needed. Frozen cells were thawed on ice and used immediately in the transformation assay. TSS consisted of PEG (polyethylene glycols, molecular weight: 3350), 5% (v/v) DMSO (dimethyl sulfoxide), and 40 mM Mg2+ (MgCl2 ), at a final pH of 6.5.
For Western blot analysis, the incubation medium of HeLahPEPT1/HeLa-hPEPT2 cells was removed and the cells were rapidly rinsed three times with 3 ml of ice-cold PBS (phosphate-buffered saline). 24-well plates that had been frozen by the ice pack and stored in RIPA (rapid immunoprecipitation assay) buffer containing protease inhibitors (proteinase inhibitors and phosphatase inhibitors). The lysate was centrifuged at 12,000 × g for 30 min to remove cell debris and collect the supernatant. Protein was measured according to the procedure of BCA (bicinchoninic acid) Kit with bovine serum albumin as the standard. Equal amounts of 10 g of proteins were resuspended in electrophoresis sample buffer containing -mercaptoethanol and separated by electrophoresis on a pre-cast 10% SDS-polyacrylamide gel (Bio-Rad, Hercules, CA), followed by electrotransfer to a PVDF membrane (Millipore, Bedford, MA). Membranes were blocked using 5% skim milk in Tris-buffered saline with 0.1% Tween-20 (TBST). -Actin served as loading control. Membranes were incubated overnight at 4 ◦ C with a 1:500 dilution of polyclonal antibody for hPEPT1/hPEPT2, and with a 1:2000 dilution of monoclonal antibody for -actin (Santa Cruz Biotechnology, Santa Cruz, CA). After washing three times in TBST, membranes were incubated for 1 h at 37 ◦ C with a 1:5000 dilution of anti-rabbit or a 1:1000 dilution of anti-mouse horseradish peroxidase-conjugated secondary antibody respectively (Invitrogen, Shanghai, China). After extensive washing with TBST, membranes were exposed to the enhanced chemiluminescenceplus reagents (ECL) according to the manufacturer’s protocol. Emitted light was documented with a BioSpectrum-410 multispectral imaging system with a Chemi HR camera 410. Protein bands were visualized and photographed under transmitted ultraviolet light. The image was used for semiquantitative measurements based on band densitometry. RIPA buffer consisted of 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS (sodium dodecyl sulfate).
2.3. Extraction and purification of plasmid DNA
2.7. Uptake studies using cell monolayers
Endo-Free plasmid Midi Kit was used to isolate plasmid DNA from bacterial cultures. Up to 200 ml of culture can be processed
The uptake of Gly-Sar and JBP485 was measured in cells grown on 24-well plates, as described previously. The composition of the
2.2. Transformation procedure
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incubation medium was as follows (in mM): 145 NaCl, 3.0 KCl, 1.0 CaCl2 , 0.5 MgCl2 and 5 d-glucose. The pH of the medium was adjusted with a solution of NaOH or HCl. The cells were preincubated for 15 min at 37 ◦ C with 1 ml of the incubation medium. After removal of the medium, the cells were incubated with 1 ml of incubation medium (pH 6.0) containing Gly-Sar or JBP485 (0.5 mM for PEPT1, 0.1 mM for PEPT2) for 3 min at 37 ◦ C. After incubation, the medium was aspirated, and the cells were rapidly rinsed three times with 3 ml of ice-cold PBS (pH 7.4) and lysed in 0.3 ml/well of PBS with 1% Triton X-100 for 60 min. The aliquots were subjected to both LC–MS/MS (liquid chromatography–mass spectrometry), as described below, and protein quantification using the BCA Kit. Uptake activity was determined as the concentration of Gly-Sar and JBP485 by LC–MS/MS per ml of protein every 3 min. To measure the Gly-Sar transport kinetics, Gly-Sar or JBP485 uptake, within a concentration range of 0.050–10 mM and 0.010–5 mM respectively, was assessed with an incubation time of 3 min. 2.8. Drug–drug interaction using cell monolayers HeLa-hPEPT1/HeLa-hPEPT2 cells were grown in DMEM with 10% FBS with antibiotics in an atmosphere of 5% CO2 /95% air at 37 ◦ C, respectively. The assays were carried out in 24-well culture plates, with nearly confluent cells seeded 48 h before each experiment. Immediately before the experiment, the cells were washed twice with 1 ml HBSS (Hank’s balanced salt solution) buffer at room temperature and then incubated with 1 ml HBSS for 15 min at 37 ◦ C. The pH of the medium was adjusted with a solution of NaOH or HCl. After removal of the medium, the cells were incubated with 1 ml HBSS (pH 5.8 for HeLa-hPEPT1 cells and pH 6.5 for HeLa-hPEPT2 cells) containing drugs including: (1) JBP485 alone (0.5 mM for HeLa-hPEPT1 cells) as control of JBP 485; (2) cephalexin alone (1 mM for HeLa-hPEPT1 cells) as control of cephalexin; (3) JBP485 (0.5 mM) + cephalexin (1 mM) for HeLa-hPEPT1 cells as the inhibitory group; (4) JBP485 alone (0.1 mM for HeLa-hPEPT2 cells) as control of JBP485; (5) lisinopril alone (0.02 mM for HeLa-hPEPT2 cells) as control of lisinopril; or (6) JBP485 (0.1 mM) + lisinopril (0.02 mM for HeLa-hPEPT2 cells) as the inhibitory group. After incubation for the designated times at 37 ◦ C with gentle shaking, the experiment was terminated by removing the medium, followed by washing three times with 1 ml ice-cold HBSS and lysed in 0.3 ml/well HBSS with 1% Triton X-100 for 60 min. The aliquots were subjected to LC–MS/MS. The uptake assays of the two agents were measured in a time-dependent manner, at the uptake time of 1, 3, 5, 10 and 15 min for HeLa-hPEPT1 cells and HeLa-hPEPT1 cells. HBSS consisted of 8 g/L NaCl, 0.4 g/L KCl, 1 g/L d-glucose, 60 mg/L KH2 PO4 , 47.5 mg/L Na2 HPO4 and 1000 ml tri-distilled water. The pH = 7.2 of the medium was adjusted with a solution of NaOH or HCl. 2.9. Biological sample preparation The concentrations of JBP485, Gly-Sar, cephalexin (CEX) and lisinopril in cell lysates were determined by LC–MS/MS. A 50-l aliquot of internal standard (IS, 500 ng/ml, JBP923 for JBP485, GlySar and cephalexin; paracetamol for lisinopril) and 500 l methanol were added to 25 l cell lysates. The mixture was vortexed for 1 min and centrifuged at 16,099 × g for 10 min to remove the protein precipitate; the upper organic layer was transferred into a polythene tube and dried with nitrogen at 37 ◦ C. The dried residue was dissolved by mobile phase, and 10 l was injected into the LC–MS/MS. 2.10. LC–MS/MS analysis An Agilent LC systems (Agilent HP1200, Agilent Technology Inc., Palo Alto, CA, USA) was used. Isocratic chromatographic separation
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was performed on a Hypersil BDS-C18 column 150 mm × 4.6 mm i.d., 5 m (Dalian Elite Analytical Instruments Co. Ltd., China). The mobile phase consisted of 3% methanol and 97% water with 0.1% formic acid for JBP 485, Gly-Sar, cephalexin and JBP923 (IS), 30% methanol and 70% water with 0.1% formic acid for lisinopril and paracetamol (IS). The flow rate was 0.5 ml/min. The column was maintained at room temperature. An API 3200 triple-quadrupole mass spectrometer (Applied Biosystems, Concord, Ont, Canada) was operated with a TurboIonspray interface in positive ion mode. Analyst 1.4.1 software (Applied Biosystems) was used for the control of equipment, data acquisition and analysis. For the optimization of MS/MS parameters, the instrument was operated with an ion spray voltage at +4.5 kV, heater gas temperature at 450 ◦ C, nebulizer gas (Gas 1) at 0.34 MPa, heater gas (Gas 2) at 0.34 MPa, curtain gas at 0.060 MPa and collision gas at 0.030 MPa. All gases used were nitrogen. Declustering potential was set at 35 V for both the analytes and IS. Multiple reaction monitoring (MRM) was employed for data acquisition. The optimized ‘truncated’ MRM fragmentation transitions were m/z 201.1 → m/z 86.1 with collision energy (CE) of 33 eV for JBP 485, m/z 147.1 → m/z 60.1 with CE of 40 eV for Gly-Sar, m/z 348.1 → m/z 174.1 with CE of 22 eV for CEX, m/z 219.1 → m/z 86.1 with CE of 38 eV for JBP923 (IS), m/z 406.1 → m/z 206.0 with CE of 30 eV for lisinopril and m/z 152.1 → m/z 110.1 with CE of 24 eV for paracetamol (IS). The dwell time for each transition was 200 ms. 2.11. Protein assay After solubilizing cells in 1% Triton X-100, 10 l of cell lysates was taken from each well for protein concentration determination using the BCA protein assay kit (Solarbio), according to the manufacturer’s instructions. Bovine serum albumin (BSA) was used as a standard. 2.12. Statistical analysis All data are given as the mean ± SD of three independent experiments. The kinetic parameters were calculated using nonlinear regression methods and confirmed by linear regression of the respective Eadie–Hofstee plots. IC50 (i.e., the concentration of the compound necessary to inhibit 50% of specific substrate carriermediated uptake) values were determined by nonlinear regression using the logistical equation for an asymmetric sigmoid (allosteric Hill kinetics): Y = Min + (Max − Min)/(1 + (X/IC50 )P ), where Max is the initial Y value, Min the final Y value, and the power P represents the Hill coefficient. Inhibition constants (Ki ) were calculated from IC50 values according to the method developed by Cheng and Prusoff: IC50 = Ki (1 + [S]/Km ) [2,7]. Statistical analysis was carried out using the SPSS11.5 package. Test results were expressed as mean ± SD. To test for statistically significant differences among multiple treatments for a given parameter, one-way analysis of variance (ANOVA) was performed. The statistical significance of differences between mean values was calculated using the non-paired t-test. If p was <0.05 or <0.01, differences were considered statistically significant. 3. Results 3.1. Transformation, extraction and purification of plasmid After transformation, plasmid was prepared from 200 ml of bacterial suspension. After extraction and purification steps, a large amount of pure plasmid DNA was obtained. The approximate concentrations of the plasmids were 0.8 mg/ml for pcDNA 3.0-hPEPT1 and 0.4 mg/ml for pcDNA 3.0-hPEPT2, as determined by agarose gel electrophoresis (Fig. 1). The pcDNA 3.0 expression vector with
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Fig. 1. Identification and semiquantitative analysis of pcDNA3-hPEPT1/-hPEPT2 by agarose gel electrophoresis. (A) pcDNA3/PEPT1 (+); (B) pcDNA3/PEPT1 (−); (C) pcDNA3/PEPT2 (+); (D) pcDNA3/PEPT2 (−).
the human PEPT1/PEPT2 cDNA inserted was transfected into HeLa cells, and approximately 0.8 mg/L of G418-resistant cells were selected and examined for Gly-Sar or JBP485 transport activities. Clones with the highest transport activities was selected (HeLahPEPT1/HeLa-hPEPT2 cells) and used for further characterization of human PEPT1/PEPT2.
3.2. Identification of HeLa-hPEPT1 and HeLa-hPEPT2 cells by Western blot analysis To confirm that the stably transfected HeLa-hPEPT1/HeLahPEPT2 cells (human PEPT1/PEPT2-expressing HeLa cells) were constructed successfully, the protein expression levels of hPEPT1 and HPEPT2 were examined by Western blot analysis. As shown in Fig. 2, PEPT1 and PEPT2 were found almost exclusive in HeLahPEPT1 and HeLa-hPEPT2 cells, respectively, but not found in the mock cell.
Fig. 2. Identification analysis of hPEPT1/hPEPT2 transfected cells by Western blot. (A) HeLa-hPEPT1 cells and (B) HeLa-hPEPT2 cells.
3.3. Characteristics of Gly-Sar or JBP485 uptake by HeLa-hPEPT1 and HeLa-hPEPT2 cells Time dependence assay. First, we examined the time course of Gly-Sar or JBP485 uptake at pH 6.0 by HeLa-hPEPT1 (human PEPT1expressing HeLa cells) and HeLa-hPEPT2 cells. The rate of Gly-Sar or JBP485 uptake in HeLa-hPEPT1 cells was much greater than that observed in HeLa-hPEPT2 cells (Fig. 3). On the other hand, the uptake of Gly-Sar or JBP485 by the parental HeLa cells transfected with control vector (HeLa-empty vector) was negligible compared with that by HeLa-hPEPT1 and HeLa-hPEPT2 cells. Linear uptakes were detected up to 3 min for HeLa-hPEPT1 cells and HeLa-hPEPT2 cells, respectively. pH dependence assay. To confirm that uptake of JBP485 and Gly-Sar by HeLa-hPEPT1 and HeLa-hPEPT2 cells was dependent on hydrogen ions, the effects of pH of the medium on Gly-Sar or JBP485 uptake by HeLa-hPEPT1 and HeLa-hPEPT2 cells were examined. In HeLa-hPEPT1 cells, Gly-Sar or JBP485 uptake was maximal at pH 5.8, whereas it was maximal at pH 6.5 in HeLa-hPEPT2 cells
Fig. 3. Time dependence of Gly-Sar or JBP485 uptake by hPEPT1-HeLa cell (A and C) and hPEPT2-HeLa cell (B and D). Cells were incubated at 37 ◦ C with Gly-Sar (A and B) or JBP485 (C and D) (A and C, 0.5 mM; B and D, 0.1 mM). Each symbol and bar represents the mean ± SD of three experiments.
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Fig. 4. pH dependence of Gly-Sar uptake by hPEPT1-HeLa cell (A) and hPEPT2-HeLa cell (B). Cells were incubated at 37 ◦ C with Gly-Sar (A, 0.5 mM; B, 0.1 mM). Each symbol and bar represents the mean ± SD of three experiments.
(Fig. 4). These data suggested uptake of JBP485 and Gly-Sar by the H+ /peptide symporter PEPT1 and PEPT2. We determined the uptake of Gly-Sar or JBP485 at pH 5.8 for HeLa-hPEPT1 cells and at pH 6.5 for HeLa-hPEPT2 cells in our subsequent experiments. Concentration dependence assay. To further confirm that our transfected models had been constructed successfully, the concentration dependence of Gly-Sar or JBP485 uptake in HeLahPEPT1/HeLa-hPEPT2 cells was determined. Specific uptake was calculated by subtracting the nonspecific uptake, which was estimated in the presence of excess unlabeled dipeptide, from the total uptake, and kinetic parameters were calculated according to the Michaelis–Menten equation. The Michaelis–Menten constant (Km ) values of Gly-Sar uptake by the hPEPT1 and hPEPT2expressing transfectants were 1.03 mM and 0.0965 mM (Fig. 5A and B, insets), respectively, and the Km values of JBP485 uptake were 1.33 mM (for PEPT1) and 0.144 mM (for PEPT2) (Fig. 5C and D, insets). These results suggested that human PEPT2 has 10-fold higher affinity for Gly-Sar or JBP485 uptake than PEPT1. The maximum velocity (Vmax ) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.86 nmol/mg protein/3 min and 0.664 nmol/mg protein/3 min, respectively, and the Vmax values of JBP485 uptake were 1.75 nmol/mg protein/3 min and 0.545 nmol/mg protein/3 min, respectively. 3.4. Mutual inhibition between Gly-Sar and JBP485 in HeLa-hPEPT1/HeLa-hPEPT2 cells To determine the targets of the interaction between Gly-Sar and JBP485, the concentration-dependent inhibition of Gly-Sar uptake
by JBP485 in the HeLa-hPEPT1 (Fig. 6A) and HeLa-hPEPT2 (Fig. 6B) cells, as well as the concentration-dependent inhibition of JBP485 uptake by Gly-Sar in the HeLa-hPEPT1 (Fig. 6C) and HeLa-hPEPT2 (Fig. 6D) cells, were investigated. The uptake of Gly-Sar was significantly inhibited by JBP485 with a Ki (inhibition constants) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). The uptake of JBP485 was also significantly inhibited by Gly-Sar with a Ki value of 12.9 mM (for PEPT1) and 2.98 mM (for PEPT2). These findings suggested that the targets of interaction between Gly-Sar and JBP485 were PEPT1 and PEPT2. 3.5. Drug–drug interaction between JBP485 and cephalexin in HeLa-hPEPT1 cells To investigate that the target of DDI between JBP485 and cephalexin was related to PEPT1 in the intestine, HeLa-hPEPT1 cells were used to examine the uptake of JBP 485 and cephalexin in the hPEPT1-transfected cells. The uptake concentration of JBP485 or cephalexin in HeLa-hPEPT1 cells was significantly reduced (Fig. 7A and B) when the two drugs were co-administered. On the other hand, the uptake of JBP485 or cephalexin by the mock cells was negligible compared with the uptake by HeLa-hPEPT1 cells. 3.6. Drug–drug interaction between JBP485 and lisinopril in HeLa-hPEPT2 cells To investigate that the target of DDI between JBP485 and lisinopril is PEPT2 in the kidneys, we investigated the uptake interaction of JBP485 and lisinopril in HeLa-hPEPT2 cells. Lisinopril markedly
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Fig. 5. Concentration dependence of Gly-Sar or JBP485 uptake by hPEPT1-HeLa cell (A and C) and hPEPT2-HeLa cell (B and D). Cells were incubated for 3 min at 37 ◦ C with Gly-Sar (A and B) or JBP485 (C and D). Each symbol and bar represents the mean ± SD of three experiments. Insets: Eadie–Hofstee plots of the uptake after correction for the nonsaturable component; V, Gly-Sar/JBP485 uptake rate (nmol/mg protein/3 min); S, Gly-Sar/JBP485 concentration (mM).
inhibited the uptake of JBP485 in HeLa-hPEPT2 cells (Fig. 8A). On the other hand, JBP485 also markedly inhibited the uptake of lisinopril in HeLa-hPEPT2 cells (Fig. 8B). The uptake of JBP485 or lisinopril was significantly greater in HeLa-hPEPT2 cells than in the mock cells.
4. Discussion Peptide transporters PEPT1 and PEPT2 have been demonstrated to play important physiological and nutritional roles in cells, and have also been shown to be of pharmacokinetic and pharmacological significance. PEPT1/PEPT2 not only serves to mediate the absorption/reabsorption of nutrients but also functions in the transport of exogenous compounds that have peptide-like structures, such as ACEIs and some -lactam antibiotics [6,12]. Therefore, when substrates of PEPT1/PEPT2 combine, their pharmacological effects may be altered. In the present study, to investigate targets in the absorption/reabsorption of these substrates, we established HeLa cells stably transfected with human PEPT1/PEPT2 cDNA and therefore expressing a proposed protoncoupled dipeptide transporter. Transporter-expressing cell models have proved to be high throughput approaches that can rapidly provide the information for identifying interaction between a compound and a particular transporter. Several attempts to develop a cell culture model having enhanced peptide transport activity have been taken by
use of cloning and recombinant DNA technology and some overexpression systems have well established. For instance, hPEPT1 has been functionally expressed in Caco-2 cells and also stably transfected into the CHO cells [4,5]. In our work, the human PEPTs transporter expressed in HeLa cells was developed. Compared to CHO cells, HeLa cells are human-derived cells which can avoid species differences. Compared to Caco-2 cells, HeLa cells have a low expression level of PEPTs, eliminating the possibility that an endogenous transporter could complicate interpretation of experiments. In our study, mock cells (empty-vector transfected HeLa cells) have little baseline uptake of Gly-Sar and JBP485 which have been improved to be a substrate of hPEPT1 and hPEPT2 in our previous experiment. There are various methods for the introduction of DNA or RNA molecules into cultured mammalian cells. These methods include chemical (liposome-mediated), physical (electroporation, microinjection, heat shock) or viral-based (retrovirus, adeno-associated virus) delivery systems. The liposome-mediated transfection method has several advantages. First, the method is simple and highly reproducible. Second, exogenous DNAs can be introduced into large numbers of cells, and third, the liposomes exhibit low cytotoxicity. To confirm that the transfected cells had been constructed successfully, we performed assays to investigate the protein expression of PEPT1/PEPT2 and the time, pH and concentration dependence of Gly-Sar and JBP485 uptake. First, by Western blot analysis of protein expression, PEPT1 and PEPT2 were highly
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Fig. 6. Interaction between Gly-Sar and JBP485 in hPEPT1-HeLa cell (A and C) and hPEPT2-HeLa cell (B and D). The uptake of Gly-Sar (A, 0.5 mM; B, 0.1 mM) was inhibited by JBP485 (A, 0.5–50 mM; B, 0.1–10 mM). The uptake of JBP485 (C, 0.5 mM; D, 0.1 mM) was inhibited by Gly-Sar (C, 0.5–50 mM; D, 0.1–10 mM). Each symbol and bar represents the mean ± SD of three experiments.
expressed in HeLa-hPEPT1 cells and HeLa-hPEPT2 cells relative to the mock cells, respectively, which confirmed that the transfected cell were constructed successfully on the protein level (Fig. 2). hPEPT1-HeLa cells were selected by measuring the uptake activities of Gly-Sar and JBP485. As shown in Fig. 3, significantly greater accumulation of Gly-Sar or JBP485 was detected in hPEPT1-HeLa
cells than in the empty vector-containing cells. Linear uptakes were recorded before 3 min. Similar results were obtained in the time dependence assays for uptake of Gly-Sar or JBP485 in hPEPT2-HeLa cells (Fig. 3B and D). Differences in the substrate affinity and pH dependence of PEPT1 and PEPT2 for Gly-Sar and JBP485 uptake suggested that these transporters play distinct roles in intestinal
Fig. 7. Time profiles of the uptake of JBP485 (A) and cephalexin (B, CEX) by HeLa–hPEPT1 cells. The time-dependent uptake of JBP485 (0.5 mM) and cephalexin (CEX, 1 mM) by HeLa–hPEPT1 cells were examined at 37 ◦ C. (Each symbol and bar represents the mean ± SD of three experiments; *p < 0.05 vs. control, **p < 0.01 vs. control.)
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Fig. 8. Time profiles of the uptake of JBP485 (A) and lisinopril (B) by HeLa–hPEPT2 cells. The time-dependent uptake of JBP485 (0.1 mM) and lisinopril (0.02 mM) by HeLa–hPEPT2 cells were examined at 37 ◦ C. (Each symbol and bar represents the mean ± SD of three experiments; *p < 0.05 vs. control, **p < 0.01 vs. control.)
absorption and tubular reabsorption of oligopeptides. The uptake of Gly-Sar or JBP485 in HeLa-hPEPT1 cells was maximal at pH 5.8, whereas it was maximal at pH 6.5 in HeLa-hPEPT2 cells. These results suggested that HeLa-hPEPT1 cells prefer a more acidic pH to transport Gly-Sar or JBP485 than HeLa-hPEPT2 cells (Fig. 4). Furthermore, the results confirmed that both hPEPT1 and hPEPT2 were proton-coupled dependence transporters. The Michaelis–Menten Km values of PEPT1 and PEPT2 for Gly-Sar uptake were similar to previously reported values (Km value of 1.1 mM in LLC-rPEPT1 cells and 0.11 mM in LLC-rPEPT2 cells for Gly-Sar) [18]. By contrast, the Km values for JBP485 uptake were found to be 1.33 mM for PEPT1 and 0.144 mM for PEPT2 in the human PEPT1- and PEPT2expressing transfectants. These findings confirm that PEPT2 is the high-affinity type transporter in the kidney, whereas PEPT1 is the low-affinity type transporter in the small intestine. Importantly, our findings revealed that human PEPT1 and PEPT2 have different characteristics, not only regarding substrate affinity for native dipeptide, but also in the recognition of a variety of peptide-like drugs. As mentioned above, the Km values for Gly-Sar uptake by HeLahPEPT1/hPEPT2 cells were significantly lower than those for JBP485 uptake. Gly-Sar is a l-l-dipeptides and has a peptide bond with trans conformation. PEPT1 is stereospecific in the sense that ll-dipeptides and l-l-l-tripeptides display a much higher affinity than dipeptides or tripeptides containing d-amino acids [24]. The structure of Gly-Sar was a peptide bond with trans conformation. A previous study investigated the influence of the substrate backbone dynamics caused by peptide bond cis/trans isomerization on the intestinal peptide transport [25]. Trans contents and affinities for PEPT1 were positively correlated. It was concluded that PEPT1 accepts trans conformers of zwitterionic dipeptides and derivatives thereof regardless of size, hydrophobicity and aromatic nature of the N-terminal amino acid. Therefore, the interaction between GlySar and the peptide transporter would be much stronger than the interaction between cyclo-dipeptide and the peptide transporter. Clinical drug–drug interactions are expected to enhance the beneficial effects of a particular drug or reduce its toxicity. However, some drug combinations will enhance the toxic effects, or reduce the pharmacologic effects; these are undesirable drug–drug interactions. 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. When JBP485 or cephalexin was used alone, the uptake concentration of JBP485 or cephalexin was significantly higher than that in the co-administered groups. This result
indicated that the target of DDI between JBP485 and cephalexin was related, at least in part, to PEPT1. The uptake of JBP485 or lisinopril was inhibited significantly when the two drugs were added simultaneously to the HeLa-hPEPT2 cells. This result indicated that the targets of DDI between JBP485 and lisinopril were, at least in part, PEPT2 in the kidneys. Our findings provide useful information regarding potential drug design strategies and delivery systems, which may be used to improve the efficiency of drug therapy. Our results may also prove important in appropriately interpreting and predicting the potential for transporter-associated DDI and toxicity. 5. Conclusion (1) Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully. (2) The functions of the transfected cells were identified using the substrates JBP485 and Gly-Sar. (3) 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. Acknowledgements This work was supported in part by a grant from the National Natural Science Foundation of China (No. 81072694) and Dalian Government (Nos. 2009E12SF155 and 2010E12SF060). We wish to express our deep gratitude to Yukio Kato (Kanazawa University, Kanazawa, Japan) for providing pcDNA3-PEPT1 and pcDNA3PEPT2. References [1] Cang J, Zhang J, Wang CY, Liu Q, Meng Q, Wand D, et al. Pharmacokinetics and mechanism of intestinal absorption of JBP485 in rats. Drug Metab Pharmacokinet 2010;25:500–7. [2] Cheng Y, Prusoff WH. Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (IC50) of an enzymatic reaction. Biochem Pharmacol 1973;22:3099–108. [3] Daniel H, Adibi SA. Transport of -lactam antibiotics in kidney brush border membrane. Determinants of their affinity for the oligopeptide/H1 symporter. J Clin Invest 1993;92:2215–23. [4] Han HK, Rhie JK, Oh DM, Saito G, Hsu CP, Stewart BH, et al. CHO/hPEPT1 cells overexpressing the human peptide transporter (hPEPT1) as an alternative in vitro model for peptidomimetic drugs. J Pharm Sci 1999;88:347–50. [5] Hsu CP, Walter E, Merkle HP, Rothen-Rutishauser B, Wunderli-Allenspach H, Hilfinger JM, et al. Function and immunolocalization of overexpressed human
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