Abstract
Carnosine (β-alanyl-l-histidine) affects a plethora of signaling pathways and genes in different biological systems. Although known as a radical scavenger, not all of these effects can simply be ascribed to its chemical nature. As previous experiments pointed towards the possibility that carnosine affects epigenetic regulation via histone acetylation, we investigated this hypothesis using the glioblastoma cell lines U87 and T98G in which carnosine’s anti-neoplastic effect is accompanied by increased expression of pyruvate dehydrogenase kinase 4. Viability and expression of PDK4 was analyzed after incubation in carnosine and different histone deacetylase inhibitors (HDACi) using cell-based assays and qRT-PCR. In addition, chromatin immunoprecipitation (ChIP) experiments were performed and the global influence of carnosine on histone H3 acetylation was analyzed by Western blot. Carnosine as well as the HDACi used increased expression of PDK4. In addition, all compounds reduced cell viability, although differences were observed with regard to magnitude and required concentrations. ChIP analysis revealed increased acetylation of histone H3 in the PDK4 promoter of U87 and T98G cells (~ 1.3- and ~ 1.7-fold, respectively) 6 h after the addition of carnosine (50 mM) followed by increased expression of PDK4 mRNA. Western blots did not detect a general increase of H3 acetylation at a genome-wide scale under the influence of carnosine. Our experiments for the first time demonstrate that carnosine influences epigenetic regulation via increased histone acetylation.
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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. We also thank Berthold Technologies (Bad Wildbad, Germany) for providing us a Mithras LB 940 Multimode Microplate Reader and last but not least Mr. Rainer Baran-Schmidt for technical assistance.
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AA and HO performed the experiments presented in the manuscript. CS performed the experiments presented in Supplement 3. HO, JM and FG supervised the experiments. All authors read and contributed to writing the manuscript. FG supervised and planned the experiments, analyzed the data together with AA and HO and wrote the manuscript.
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Oppermann, H., Alvanos, A., Seidel, C. et al. Carnosine influences transcription via epigenetic regulation as demonstrated by enhanced histone acetylation of the pyruvate dehydrogenase kinase 4 promoter in glioblastoma cells. Amino Acids 51, 61–71 (2019). https://doi.org/10.1007/s00726-018-2619-2
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DOI: https://doi.org/10.1007/s00726-018-2619-2