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  • br The efficacy of BNCT is

    2020-08-18


    The efficacy of BNCT is highly dependent on the selective de-livery and accumulation of 10B into tumors. Although various boron delivery agents Gefitinib have been synthesized,5 most of them have been evaluated only in preclinical models. Only two boron delivery agents are currently adopted in clinical, BSH (disodium mercapto-closo-undecahydrododecaborate [B12H11SH]2-2Naþ) and BPA
    1347-8613/© 2019 The Authors. Production and hosting by Elsevier B.V. on behalf of Japanese Pharmacological Society. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
    (p-borono-L-phenylalanine).6,7 BSH has been used for the treatment of high grade gliomas. Its tumor-targeting is dependent on the disrupted blood-brain barrier in glioma and subsequent passive diffusion into tumor cells.8 In contrast, BPA is delivered into tumor cells by transporter-mediated uptake. We have recently demon-strated that BPA-uptake into tumor cells is mediated by cancer-upregulated amino Gefitinib transporters LAT1 and ATB0,þ.9
    The usefulness of cancer-upregulated transporters in the de-livery of boron compounds into tumor cells has provided us with the further possibility to design new boron compounds for BNCT tar-geting other cancer-upregulated transporters. In tumor cells, it has been shown that not only glucose and amino acid transporters but also the transporters for oligopeptides are upregulated due to increased metabolic requirement.10 It was proposed that the oligo-peptide transporters in tumor cells could be used as a tool for the delivery of anti-tumor agents into tumor cells.10 Previous studies have suggested that Hþ-coupled oligopeptide transporters, PEPT1 and/or PEPT2, are expressed in various types of tumor cell lines including those of hepatocarcinoma,11 pancreatic carcinoma,12 gastric cancer,13 and prostate cancer.14 mRNAs of PEPT1 and PEPT2 were reported to be detected in 27.6% and 93.1% of the examined 58 human tumor cell lines, respectively.15 We previously synthesized
    dipeptides of BPA and tyrosine, BPA-Tyr (p-L-BPA-L-tyrosine) and Tyr-BPA (L-tyrosine-p-L-BPA),16,17 though their potential as boron
    delivery agents has not been evaluated. In this study, we have pur-sued the possibility of oligopeptide transporter-targeted boron de-livery into tumor cells by using the BPA-containing dipeptides.
    2. Materials and methods
    Tritium-labeled glycylsarcosine ([3H]Gly-Sar) (7.4 GBq/mmol) was from Moravek Biochemicals Inc. (Brea, CA, USA). Unlabeled glycylsarcosine (Gly-Sar) was from MP Biomedicals (Santa Ana, CA, USA). EMEM, RPMI-1640, and DMEM were from SigmaeAldrich (St Louis, MO, USA). Ham's F-12 medium was from Wako Pure Chem-icals (Osaka, Japan). PNGaseF was from New England Bio-laboratories (Ipswich, MA, USA). BPA-Tyr and Tyr-BPA (Fig. 1) were synthesized as described previously.16
    Details of cell lines used in the study are available in Table S1. Cells were grown at 37 C in a humidified atmosphere with 5% CO2. Media were supplemented with 10% heat-inactivated FBS (Gibco Life Technologies, Grand Island, NY, USA), 100 units/mL penicillin, and 100 mg/mL streptomycin (Nacalai Tesque, Kyoto, Japan).
    2.3. Establishment of HEK293 cells stably expressing PEPT1 or PEPT2
    cDNA was generated from human kidney total RNA (Clontech, Palo Alto, CA, USA) by reverse transcription using SuperScriptIII First-Strand Synthesis for RT-PCR (Invitrogen, San Diego, CA, USA). The full-length coding sequence of PEPT1 was amplified by PCR, using the following primer pair: forward 50-gggcttaaggccaccatgg-gaatgtccaaatcacacagtt-30 and reverse 50-gggtctagatcacatctgtttctgtg aattggcc-30. The obtained PCR product was cloned into pcDNA 3.1(þ) plasmid (Invitrogen) at AflII/XbaI restriction enzyme sites to obtain pcDNA3.1(þ)-PEPT1. pcDNA3.1(þ)-PEPT2 was constructed as described previously.18 Stable transfectants of HEK293 cells for pcDNA3.1(þ)-PEPT1 (HEK293-PEPT1 cells), pcDNA3.1(þ)-PEPT2 (HEK293-PEPT2 cells), and empty pcDNA3.1(þ) (MOCK cells) were established as described previously.19 
    OH
    O
    N COOH
    HO
    H
    B NH2
    BPA-Tyr
    OH OH
    B
    OH
    O
    NCOOH
    H NH2 HO Tyr-BPA
    Fig. 1. BPA-containing dipeptides used in the present study.
    uptake of [3H]Gly-Sar was examined at pH 6.0 optimal for Hþ-coupled oligopeptide transport.21,22 Then, the cells were lysed and the radioactivity was measured by liquid scintillation counting. The protein concentration of cell lysate was determined by Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). For inhibition experiments, the indicated concentrations of BPA-containing dipeptides dissolved in DMSO were added. The IC50 values were determined by measuring the uptake for 5 min in the presence of indicated concentrations of BPA-Tyr or Tyr-BPA. To measure the inhibitory constant (Ki), the [3H]Gly-Sar uptake at indicated concentrations was measured for 5 min in the presence or absence of BPA-Tyr or Tyr-BPA. Nonlinear regression analyses using SigmaPlot 12.5 (Systat Software, Inc. Chicago, IL, USA) were performed as described previously.20