Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Kansas City, Missouri 64108, United States.
Mol Pharm. 2013 Feb 4;10(2):477-87. doi: 10.1021/mp300364k. Epub 2012 Sep 21.
Peptide transporters are expressed predominantly in intestinal and renal epithelial cells. The functional expression of peptide transporters is also identified in other types of tissues, such as glia cells, macrophages, and the epithelia of the bile duct, the lungs, and the mammary glands. However, their presence and role are poorly understood in carcinomas. We explored the expression profile and functional activity of peptide transporters in the prostate cancer cell lines LNCaP, PC-3, and DU145. Quantitative real time RT-PCR (qRT-PCR) and Western blot were used to evaluate the expression profile of peptide transporter 1 (PEPT1), peptide transporter 2 (PEPT2), peptide histidine transporter 1 (PHT1), and peptide histidine transporter 2 (PHT2) in these cells. LNCaP expresses high levels of PEPT2 and PHT1, while PC-3 demonstrates strong expression of PEPT1 and PHT1. DU145 shows only weak expression of PEPT1 and PHT1. Functional activities were studied in these cell lines using radiolabeled glycylsarcosine ([(3)H]Gly-Sar) and l-histidine ([(3)H]-l-histidine). The uptake of [(3)H]Gly-Sar and [(3)H]-l-histidine was time- and pH-dependent. A kinetic study showed that the uptake of Gly-Sar and l-histidine is saturable over the tested concentration range. The binding affinity (K(m)) and the maximal velocity (V(max)) exhibited in the three cell lines were consistent with the expression profiles we observed in qRT-PCR and Western blot analysis. A competitive inhibition study revealed that peptide transporters in prostate cancer cells exhibited broad substrate specificity with a preference for hydrophobic dipeptides, such as Leu-Leu. Fluorescence microscopy study revealed that the fluorescent dipeptide probe d-Ala-Lys-AMCA (a substrate of peptide transporters) specifically accumulated in the cytoplasm of LNCaP and PC-3, but not DU145 cells. Inhibiting the peptide transporter activity by Gly-Sar suppressed the growth of LNCaP and PC-3 cells. Our study indicated that PC-3 cells can be established as a new cell culture model for PEPT1 study, and LNCaP can be used as a model for PEPT2 study. Moreover, our results suggested that peptide transporters are overexpressed in prostate cancer cells and can be adopted as a promising target for tumor-specific drug delivery.
肽转运体主要在肠道和肾脏上皮细胞中表达。肽转运体的功能表达也在其他类型的组织中被鉴定出来，如神经胶质细胞、巨噬细胞、胆管、肺和乳腺的上皮细胞。然而，它们在癌中的存在和作用还知之甚少。我们探索了肽转运体在前列腺癌细胞系 LNCaP、PC-3 和 DU145 中的表达谱和功能活性。定量实时 RT-PCR（qRT-PCR）和 Western blot 用于评估这些细胞中肽转运体 1（PEPT1）、肽转运体 2（PEPT2）、肽组氨酸转运体 1（PHT1）和肽组氨酸转运体 2（PHT2）的表达谱。LNCaP 表达高水平的 PEPT2 和 PHT1，而 PC-3 则表现出强烈的 PEPT1 和 PHT1 表达。DU145 仅表现出弱的 PEPT1 和 PHT1 表达。使用放射性标记的甘氨酰肌氨酸（[(3)H]Gly-Sar）和 l-组氨酸（[(3)H]-l-histidine）在这些细胞系中研究了功能活性。[(3)H]Gly-Sar 和 [(3)H]-l-histidine 的摄取是时间和 pH 依赖性的。动力学研究表明，甘氨酰肌氨酸和 l-组氨酸的摄取在测试的浓度范围内是饱和的。在三种细胞系中观察到的结合亲和力（K(m)）和最大速度（V(max)）与我们在 qRT-PCR 和 Western blot 分析中观察到的表达谱一致。竞争性抑制研究表明，前列腺癌细胞中的肽转运体表现出广泛的底物特异性，偏爱疏水性二肽，如 Leu-Leu。荧光显微镜研究表明，荧光二肽探针 d-Ala-Lys-AMCA（肽转运体的底物）特异性积累在 LNCaP 和 PC-3 的细胞质中，但不在 DU145 细胞中。通过甘氨酰肌氨酸抑制肽转运体活性抑制了 LNCaP 和 PC-3 细胞的生长。我们的研究表明，PC-3 细胞可以作为研究 PEPT1 的新细胞培养模型建立，而 LNCaP 可以作为研究 PEPT2 的模型。此外，我们的结果表明，肽转运体在前列腺癌细胞中过度表达，可以作为肿瘤特异性药物输送的有前途的靶标。
