Delivery of peptide-mimetic drugs to tumors utilizing oligopeptide transporters

在本研究中，我们研究了利用在一些肿瘤细胞系中表达的寡肽转运体进行药物输送的可行性。在本研究期间，我们获得了以下研究结果。将hPEPT1基因转入HeLa细胞，建立了稳定高表达人寡肽转运蛋白PEPT1的细胞系。经甘氨酰肌氨酸测定证实其转运活性，命名为HeLa-hPEPT1.2细胞系。接种HeLa-hPEPT1的裸鼠(Balb/c nu/nu小鼠)在数周内裸露肿瘤。在这些小鼠中，静脉注射。注射抗肿瘤药物倍他汀后，hPEPT1表达的肿瘤细胞内有二肽肌肽积聚。体外MTT法显示HeLa-hPEPT1细胞对倍他汀的敏感性高于亲本HeLa细胞。口服贝斯汀对HeLa-hPEPT1裸鼠体内Tiimor生长有明显的抑制作用，而对HeLa接种小鼠的生长无明显影响。用实时定量聚合酶链式反应(Real Time PCR)定量分析了已知的寡肽转运蛋白PEPT1和PEPT2在虹膜视觉25 Tumbf细胞中的信使RNA表达水平。其中，ML-1、Nakajima和Caco-2细胞高表达PEPT1，而PEPT2在大多数细胞系中表达。比较mRNA表达和转运蛋白活性，我们发现它们之间几乎没有相关性。因此，推测存在代表多肽摄取的新型转运蛋白。对有机阳离子转运蛋白OCTN和OATPs的CDNA进行了分子克隆，并在肿瘤细胞系中进行了表达。我们已经证明了在肿瘤生长中起作用的氨基酸转运蛋白LAT1和LAT2在血脑屏障中的表达。因此，对肿瘤中表达的多种转运蛋白的详细表征将为我们建立肿瘤特异性给药方法提供有价值的信息。

In the present study, we studied feasibility of drug delivery using oligopeptide transporter which expressed in some tumor cell lines. During this study period, we obtained following findings.1. A stable cell line which over expresses human oligopeptide transporter PEPT1 was established by transfecting hPEPTl cDNA into HeLa cells. Its transport actiyity was confirmed by measuring glycylsarcosine and termed the cell line HeLa-hPEPT1.2. Nude mice (Balb/c nu/nu mice) inoculated with HeLa-hPEPT1 bared tumor within several weeks. In these mice, I.v. injected an anti-tumor drug bestatine, and dipeptide carnosine were accumulated in the hPEPT1-expressing tumors.3. HeLa-hPEPT1 was more sensitive to bestatine than parent HeLa cell line by in vitro MTT assay. Tiimor growth of HeLa-hPEPT1 in nude mice was strongly suppressed by oral dose of bestatine to the mice, while that of HeLa inoculated mice was not affected.4. Messenger RNA expression levels of known oligopeptide transporters that is PEPT1 and PEPT2 were quantitatively analyzed by the real time PCR in iridivisual 25 tumbf lines. Among them ML-1, Nakajima and Caco-2 cells expressed PEPT1in high level on the other hand, PEPT2 expression was observed majority of cell lines examined. Comparing mRNA expression and transport activity profiles, we found little correlation between them. Therefore, existence ofnovel transporter that represents the uptake of peptides has been suggested.5. Molecular cloning of CDNA of organic cation transporter OCTNs and OATPs were performed and some of which were expressed in tumor cell lines.6. We have proved that expression of amino acid transporter LAT1 and LAT2, which play roles in tumor growth, in the blood-brain barrier.In conclusion, detailed characterization of many kinds of transporters which expressed in tumors will provide us valuable information to establish method for tumor specific delivery of drugs.