Abstract
The previous studies demonstrated that carnosine (β-alanyl-l-histidine) inhibits the growth of tumor cells in vitro and in vivo. Considering carnosine for the treatment of glioblastoma, we investigated which proton-coupled oligopeptide transporters (POTs) are present in glioblastoma cells and how they contribute to the uptake of carnosine. Therefore, mRNA expression of the four known POTs (PEPT1, PEPT2, PHT1, and PHT2) was examined in three glioblastoma cell lines, ten primary tumor cell cultures, in freshly isolated tumor tissue and in healthy brain. Using high-performance liquid chromatography coupled to mass spectrometry, the uptake of carnosine was investigated in the presence of competitive inhibitors and after siRNA-mediated knockdown of POTs. Whereas PEPT1 mRNA was not detected in any sample, expression of the three other transporters was significantly increased in tumor tissue compared to healthy brain. In cell culture, PHT1 expression was comparable to expression in tumor tissue, PHT2 exhibited a slightly reduced expression, and PEPT2 expression was reduced to normal brain tissue levels. In the cell line LN405, the competitive inhibitors β-alanyl-l-alanine (inhibits all transporters) and l-histidine (inhibitor of PHT1/2) both inhibited the uptake of carnosine. SiRNA-mediated knockdown of PHT1 and PHT2 revealed a significantly reduced uptake of carnosine. Interestingly, despite its low expression at the level of mRNA, knockdown of PEPT2 also resulted in decreased uptake. In conclusion, our results demonstrate that the transporters PEPT2, PHT1, and PHT2 are responsible for the uptake of carnosine into glioblastoma cells and full function of all three transporters is required for maximum uptake.
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Acknowledgements
We like to thank Flamma [Flamma s.p.a. Chignolo d’Isola, Italy (http://www.flammagroup.com)] for the generous supply with very high-quality carnosine for all of our experiments. In addition, we like to thank Dr. Hans-Heinrich Foerster from the Genolytic GmbH (Leipzig, Germany) for genotyping and confirmation of cell identity and last not least Mr. Rainer Baran-Schmidt for technical assistance.
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MH and HO performed the experiments. CB and HO established the HPLC–MS method and performed the HPLC–MS measurements. JM did the surgery and revised the manuscript. HO and FG designed and coordinated the experiments. FG, MH, and HO wrote the manuscript. All authors read and approved the manuscript.
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Supplementary material 1 Comparison of expression of POTs in human tissues. The expression of individual transporters in percent with regard to the expression of all reporters (set as 100%) was calculated using the data from Fagerberg et al., (Fagerberg et al. 2014) which is available as FPKM (fragments per kilobase of exon model per million mapped reads) (JPEG 2457 kb)
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Supplementary material 2 Primers used for qRT-PCR. Indicated are the target genes and the sequences of the employed forward and reverse primers (DOCX 14 kb)
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Oppermann, H., Heinrich, M., Birkemeyer, C. et al. The proton-coupled oligopeptide transporters PEPT2, PHT1 and PHT2 mediate the uptake of carnosine in glioblastoma cells. Amino Acids 51, 999–1008 (2019). https://doi.org/10.1007/s00726-019-02739-w
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DOI: https://doi.org/10.1007/s00726-019-02739-w