br Uptake of BPA Tyr and

3.3. Uptake of BPA-Tyr and Tyr-BPA mediated by oligopeptide transporters
The competitive inhibition of [3H]Gly-Sar uptake by BPA-containing dipeptides strongly suggested that they are transport-able substrates of oligopeptide transporters. We, thus, examined the transport of BPA-Tyr and Tyr-BPA by measuring the boron fluxes. As shown in Fig. 3, the boron contents determined by ICP-AES in HEK293-PEPT1 and HEK293-PEPT2 Poly(I:C) after the treatment with BPA-Tyr and Tyr-BPA were significantly higher than that in MOCK cells, indicating that BPA-Tyr and Tyr-BPA were recognized as sub-strates and taken up into cells via PEPT1 and PEPT2. Boron accu-mulation was considerably higher in HEK293-PEPT1 cells than that in HEK293-PEPT2 cells. This observation, together with the higher Ki values for PEPT1 compared with PEPT2, is consistent with the property of PEPT1 as a low affinity and high capacity oligopeptide transporter.24 Tyr-BPA showed ~2-fold higher boron incorporation into HEK293-PEPT1 cells than BPA-Tyr.
3.4. Expression and function of oligopeptide transporters in tumor cells
To examine whether tumor cells functionally express PEPT1 and PEPT2, [3H]Gly-Sar uptake in various tumor cell lines (Fig. 4A) was compared with the protein expression of PEPT1 and PEPT2 evalu-ated by western blot analysis (Fig. 4B). In this study, tumor cell lines showing various level of expression of PEPT1 and PEPT2 mRNAs were selected based on the gene expression database (Cancer Cell Line Encyclopedia, Broad Institute) (Fig. S3).25 As shown in Fig. 4A and B, the level of [3H]Gly-Sar uptake in tumor cell lines well correlated with that of protein expression of PEPT1. In contrast, PEPT2 protein was not detected in any examined tumor cell lines (Fig. 4B), although high level of PEPT2 mRNA was reported for some
PEPT1
uptake 80
Control -Tyr -BPA -Sar BPA Tyr Gly (C) 
PEPT2
uptake 80
Control -Tyr -BPA -Sar BPA Tyr Gly (D)
PEPT1
PEPT1
uptake
of the cell lines in the gene expression database (Fig. S3). These observations indicate that PEPT1 but not PEPT2 is functionally expressed in the tumor cell lines examined.
Table 1 Ki values of BPA-Tyr and Tyr-BPA for oligopeptide transporters PEPT1 and PEPT2.
Compound Ki for PEPT1 (mM) Ki for PEPT2 (mM)
3.5. Incorporation of BPA-Tyr and Tyr-BPA into tumor cells via PEPT1
To assess the transport of BPA-Tyr and Tyr-BPA by PEPT1 endogenously expressed in tumor cells (Fig. 4B), the inhibitory effect of BPA-containing dipeptides on the [3H]Gly-Sar uptake of tumor cells were first examined. Three cell lines, AsPC-1, VMRC-RCW, and SUIT-2, showing high [3H]Gly-Sar uptake were selected for this study among the tumor cell lines shown in Fig. 4. As shown in Fig. 5, the [3H]Gly-Sar uptakes in these cell lines were strongly
PEPT2
MOCK
MOCK
Boronconcentration(pg/mgprotein) 200
Boronconcentration(pg/mgprotein) 20
-Tyr
-BPA
-Tyr
-BPA
BPA
Tyr
BPA
Tyr
Fig. 3. Boron accumulation in HEK293-PEPT1 and HEK293-PEPT2 cells by the treatment with BPA-Tyr and Tyr-BPA. (A) Boron contents in HEK293-PEPT1 cells (open columns) and MOCK cells (closed columns) were measured by ICP-AES after the treatment with 500 mM BPA-Tyr (left) and 500 mM Tyr-BPA (right) for 10 min. (B) Boron contents in HEK293-PEPT2 cells (open columns) and MOCK cells (closed columns) were measured by ICP-AES after the treatment with 500 mM BPA-Tyr (left) and 500 mM Tyr-
NUGC-4AsPC-1VMRC-RCW
Detroit562BICR6PC-3 AU565OVMANA SUIT-2MOCKPEPT1PEPT2
uptake 80
anti-PEPT1 (Short exposure) anti-PEPT1
(Long exposure)
anti-PEPT2
Fig. 4. [3H]Gly-Sar transport activity and expression of oligopeptide transporters in tumor cell lines. (A) [3H]Gly-Sar uptake (10 mM) was measured for 10 min in the indicated cell lines. For comparison, the [3H]Gly-Sar uptake in HEK293-MOCK, HEK293-PEPT1, and HEK293-PEPT2 cells was also measured. The values were normalized by the [3H]Gly-Sar uptake in HEK293-PEPT1 cells and expressed as “% control”. Data shown are means ± S.E.M. n ¼ 4. (B) Western blot analysis of PEPT1, PEPT2 and Naþ/Kþ ATPase a1 were conducted using the crude membrane fraction of the indicated cell lines. Samples were treated with PNGaseF. Naþ/Kþ ATPase a1 was detected as a loading control. HEK293-PEPT1 and HEK293-PEPT2 cells were used as positive controls.