br The competitive inhibition of H Gly Sar uptake by
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 ZD1839 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
Control -Tyr -BPA -Sar BPA Tyr Gly (C)
Control -Tyr -BPA -Sar BPA Tyr Gly (D)
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
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-
Detroit562BICR6PC-3 AU565OVMANA SUIT-2MOCKPEPT1PEPT2
anti-PEPT1 (Short exposure) anti-PEPT1
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. inhibited by BPA-Tyr and Tyr-BPA, suggesting that they are trans-ported by intrinsic tumor cell PEPT1.
The incorporation of BPA-Tyr and Tyr-BPA into tumor cells by PEPT1 was confirmed by siRNA-mediated knockdown in AsPC-1 cells. AsPC-1 cell line was chosen because it was used in a previous study to evaluate the uptake of [11C]Gly-Sar, a PEPT1-targeted PET tracer, in tumor xenograft model.15 When the PEPT1 expression was suppressed (Fig. 6A), the uptake of [3H]Gly-Sar was reduced signif-icantly (50~60% decrease compared with control, Fig. 6B), finally confirming the significant contribution of PEPT1 to the oligopeptide uptake of tumor cells. Consistently, boron incorporation measured with ICP-AES after the treatment with BPA-Tyr (Fig. 6C) and Tyr-BPA (Fig. 6D) was significantly reduced by PEPT1 knockdown, confirming the PEPT1-mediated uptake of BPA-containing dipeptides in tumor cells. It is noted that Tyr-BPA exhibited ~2-fold higher boron incor-poration into AsPC-1 cells compared with BPA-Tyr, which is similar to the observation in HEK293-PEPT1 cells (Fig. 3A).