ZD1839 br Inductively coupled plasma atomic emission
2.5. Inductively coupled plasma atomic emission spectroscopy (ICP-AES)
Cells were seeded on type-I collagen-coated 100 mm dishes at 9.0 105 cells/dish and cultured for 48 h. Cells were treated as described above, except that 500 mM of BPA-Tyr or Tyr-BPA was added to Naþ-free HBSS. After incubation for 10 min, cells were washed three times with Naþ-free HBSS and lysed with 1 mL of distilled water. An aliquot of the lysate was used to determine the protein concentration. The remaining samples were digested with 5 mL of 60% nitric ZD1839 (Nacalai Tesque), heated with
microwave (ETHOS1600, Milestone, Italy) for 25 min, and intro-duced to OPTIMA 8300 ICP-AES (PerkineElmer, Boston, MA, USA) to measure the boron-specific emission at 249.773 nm. Standard curves were drawn by boron standard solution (Kanto Kagaku, Tokyo, Japan).
2.6. Gene silencing by RNA interference
AsPC-1 cells were seeded at 1.0 105 cells/well on poly-D-lysine coated 24-well plates 24 hr prior to siRNA transfection. Negative control siRNA (SigmaeAldrich, MISSION siRNA Universal Negative Control #2), PEPT1 siRNA #1 (SigmaeAldrich, MISSIONsiRNA: Hs_SLC15A1_7227_s), and PEPT1 siRNA #2 (SigmaeAldrich, MIS-SIONsiRNA: Hs_SLC15A1_7228_s) were transfected with Lipofect-amine RNAiMAX (Invitrogen). The cells were used for analyses at 48 hr after transfection.
2.7. Western blot analysis
AsPC-1 cells (5.0 106 cells) mixed with the same volume of Matrigel (Corning, Corning, NY, USA) were subcutaneously injected into the back of BALB/c-nu/nu mice (female, 5-week-old). Mice were maintained as previously described.23 Three weeks after xenotransplantation, BPA-Tyr (370 mg/kg, dissolved in 200 mL of PBS) was intravenously injected. Mice were deeply anesthetized by inhaled approximately 2% isoflurane and dissected at 3 and 5 hr after BPA-Tyr administration. The collected blood and tissues were subjected to ICP-AES. The experiments were conducted according to the regulations of the Animal Care and Use Committee of Osaka University.
2.9. Statistical analysis
Unpaired Student t-test was used to compare two sets of data. One-way ANOVA with Dunnett's post-test was used for compari-sons of more than two sets of data. Differences were considered to be significant if the P-value was <0.05.
3.1. Functional expression of oligopeptide transporters in stable cell lines
The protein expression of PEPT1 and PEPT2 in the constructed stable cell lines (HEK293-PEPT1 and HEK293-PEPT2 cells) was confirmed by western blot analysis as shown in Fig. S1. The [3H]Gly-Sar uptake detected in both cell lines was considerably higher than that of MOCK cells (Fig. S1), indicating that PEPT1 and PEPT2 were functionally expressed. Uptakes of [3H]Gly-Sar almost linearly increased within 5 and 10 min of incubation time for PEPT1 and PEPT2, respectively. In all the subsequent inhibition and kinetic analyses, the uptake was measured for 5 min to determine the initial transport rates. 3.2. Interaction of BPA-Tyr and Tyr-BPA with oligopeptide transporters
Inhibition experiments were carried out to examine whether the synthesized BPA-containing dipeptides interact with oligo-peptide transporters. The uptakes of [3H]Gly-Sar medicated by PEPT1 and PEPT2 were strongly inhibited by BPA-Tyr and Tyr-BPA (Fig. 2A, B). The inhibitory effect of BPA-containing di-peptides on the [3H]Gly-Sar uptake was concentration dependent (Fig. 2CeF). The IC50 values of BPA-Tyr to inhibit PEPT1- and PEPT2-mediated uptakes of [3H]Gly-Sar (10 mM) were deter-mined to be 112.7 ± 1.2 mM and 11.5 ± 1.2 mM, respectively. The IC50 values of Tyr-BPA for PEPT1- and PEPT2-mediated uptakes of [3H]Gly-Sar (10 mM) were 801.7 ± 1.6 mM and 49.0 ± 1.1 mM, respectively.
To further characterize the inhibition profiles, the concentra-tion dependence of [3H]Gly-Sar uptakes in HEK293-PEPT1 and HEK293-PEPT2 cells was measured in the presence or absence of BPA-containing dipeptides to determine the Ki values (Fig. S2). Nonlinear regression analysis of the obtained results revealed that [3H]Gly-Sar uptakes mediated by PEPT1 and PEPT2 were competitively inhibited by BPA-containing dipeptides. Consis-tently, the double reciprocal plots indicated the apparent increase of Km in the presence of BPA-containing dipeptides without changing Vmax. The Ki values of BPA-Tyr and Tyr-BPA for PEPT1 and PEPT2 determined by nonlinear regression analysis are summa-rized in Table 1. For both PEPT1 and PEPT2, BPA-Tyr showed several times higher affinities compared with Tyr-BPA. Regarding the selectivity between the oligopeptide transporters, both BPA-Tyr and Tyr-BPA exhibited about 20e30-fold higher affinities for PEPT2 than for PEPT1.
3.3. Uptake of BPA-Tyr and Tyr-BPA mediated by oligopeptide transporters