Abstract
The naturally occurring dipeptide carnosine (β-alanyl-l-histidine) inhibits the growth of tumor cells. As its component l-histidine mimics the effect, we investigated whether cleavage of carnosine is required for its antineoplastic effect. Using ten glioblastoma cell lines and cell cultures derived from 21 patients suffering from this malignant brain tumor, we determined cell viability under the influence of carnosine and l-histidine. Moreover, we determined expression of carnosinases, the intracellular release of l-histidine from carnosine, and whether inhibition of carnosine cleavage attenuates carnosine’s antineoplastic effect. We observed a significantly higher response of the cells to l-histidine than to carnosine with regard to cell viability in all cultures. In addition, we detected protein and mRNA expression of carnosinases and a low but significant release of l-histidine in cells incubated in the presence of 50 mM carnosine (p < 0.05), which did not correlate with carnosine’s effect on viability. Furthermore, the carnosinase 2 inhibitor bestatin did not attenuate carnosine’s effect on viability. Interestingly, we measured a ~ 40-fold higher intracellular abundance of l-histidine in the presence of 25 mM extracellular l-histidine compared to the amount of l-histidine in the presence of 50 mM carnosine, both resulting in a comparable decrease in viability. In addition, we also examined the expression of pyruvate dehydrogenase kinase 4 mRNA, which was comparably influenced by l-histidine and carnosine, but did not correlate with effects on viability. In conclusion, we demonstrate that the antineoplastic effect of carnosine is independent of its cleavage.
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Acknowledgements
We would like to thank Flamma [Flamma s.p.a. Chignolo d’Isola, Italy (https://www.flammagroup.com)] for the generous supply with very high-quality carnosine for all of our experiments. In addition, we would like to thank Dr. Hans-Heinrich Foerster from the Genolytic GmbH (Leipzig, Germany) for genotyping and confirmation of cell identity and last not least Mrs. Susan Billig for technical assistance.
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KP performed most of the experiments with contributions of HO and RB-S. CB established the HPLC-MS method with contributions of HO and performed the HPLC-MS measurements. JM did the surgery and revised the manuscript. HO and FG designed the study and wrote the manuscript. All authors read and approved the manuscript.
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Supplementary material 1 (JPG 3325 kb) Supplemental Figure 1a to e: Viability of 21 primary glioblastoma cell cultures and 10 glioblastoma cell lines under the influence of carnosine and L-histidine. Glioblastoma cell cultures and cell lines were exposed for 48 hours to different concentrations of carnosine (0, 10, 25, 50 or 75 mM) or L-histidine (0, 10, 25 or 50 mM). Viability was determined by assessing metabolic activity (CTB) and by measuring the amount of ATP in cell lysates (CTG) (all in sixtuplicate). The summary of the experiments is presented in the Box blot in Fig. 1 of the main text
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Supplementary material 2 (JPG 3098 kb) Supplemental Figure 2. Cell viability under the influence of bestatin in the absence and presence of carnosine. Cells from the lines G55T2 and LN405 in the presence and absence of 50 mM carnosine were exposed to different concentrations of bestatin (0, 10, 50 or 100 µM) and the viability of cells was determined setting the viability without the inhibitor for each condition to 100 percent. Viability was determined after 24, 48 and 72 hours by measuring metabolic activity (dehydrogenase (DH) activity) and by determining the amount of ATP in cell lysates (ATP in cell lysates). Data is represented as mean and standard deviation of six independent measurements. Statistical significance was determined by Welch’s t-test with: *: p<0.05; **: p<0.005; ***: p<0.0005
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Supplementary material 3 (JPG 1975 kb) Supplemental Figure 3. Comparison of viability in the presence of 50 mM carnosine and the relative change of the intracellular abundance of L-histidine after exposure to the dipeptide. Viability of cells exposed for 48 hours to 50 mM carnosine was compared to the amount of L-histidine determined 24 hours after exposure to 50 mM of the dipeptide relative to untreated control cells (set as 100 percent). Note: For readability, error bars were omitted from the graph
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Oppermann, H., Purcz, K., Birkemeyer, C. et al. Carnosine’s inhibitory effect on glioblastoma cell growth is independent of its cleavage. Amino Acids 51, 761–772 (2019). https://doi.org/10.1007/s00726-019-02713-6
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DOI: https://doi.org/10.1007/s00726-019-02713-6