Delivery of peptide-mimetic drugs to tumors utilizing oligopeptide transporters

在本研究中，我们研究了利用在某些肿瘤细胞系中表达的寡肽转运体给药的可行性。在此研究期间，我们获得了以下发现：将人寡肽转运体PEPT1 cDNA转染HeLa细胞，建立了过表达人寡肽转运体PEPT1的稳定细胞系。通过测定甘氨酸肌氨酸证实其转运活性，并命名为细胞系HeLa-hPEPT1.2。裸鼠（Balb/c nu/nu小鼠）在几周内接种HeLa-hPEPT1裸鼠肿瘤。在这些小鼠中，静脉注射抗肿瘤药物贝司他汀，二肽肌肽在表达hpept1的肿瘤中积累。体外MTT试验表明，HeLa- hpept1对贝司他汀的敏感性高于亲本HeLa细胞株。3 .口服贝司他汀对裸鼠体内HeLa- hpept1的生长有明显抑制作用，而对接种HeLa的小鼠没有影响。采用实时荧光定量PCR技术，定量分析了虹膜25个tumbf系中已知寡肽转运体PEPT1和PEPT2的信使RNA表达水平。其中ML-1、Nakajima和Caco-2细胞高水平表达pept1，而大多数细胞系均表达PEPT2。比较mRNA表达和运输活性谱，我们发现它们之间几乎没有相关性。因此，存在一种代表肽摄取的新型转运体。对有机阳离子转运体OCTNs和OATPs的CDNA进行了分子克隆，其中部分在肿瘤细胞系中表达。我们证实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。总之，详细表征多种在肿瘤中表达的转运蛋白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.