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Amino Acids

pp 1–12 | Cite as

Carnosine’s inhibitory effect on glioblastoma cell growth is independent of its cleavage

  • Henry OppermannEmail author
  • Katharina Purcz
  • Claudia Birkemeyer
  • Rainer Baran-Schmidt
  • Jürgen Meixensberger
  • Frank Gaunitz
Original Article
  • 69 Downloads

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.

Keywords

Carnosine l-Histidine Glioblastoma Carnosinase Pyruvate dehydrogenase kinase 4 

Notes

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.

Author contributions

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.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no potential conflict of interest.

Informed consent

All patients provided written informed consent according to German law as confirmed by the local committee (#144-2008) in accordance with the 1964 Helsinki declaration and its later amendments.

Supplementary material

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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 1 (JPG 3338 kb)
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Supplementary material 1 (JPG 2799 kb)
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Supplementary material 1 (JPG 3372 kb)
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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

References

  1. Baraniuk JN, El-Amin S, Corey R, Rayhan R, Timbol C (2013) Carnosine treatment for gulf war illness: a randomized controlled trial. GJHS 5:69.  https://doi.org/10.5539/gjhs.v5n3p69 CrossRefGoogle Scholar
  2. Bellia F, Vecchio G, Rizzarelli E (2014) Carnosinases, their substrates and diseases. Molecules 19:2299–2329.  https://doi.org/10.3390/molecules19022299 CrossRefGoogle Scholar
  3. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B (Methodological) 57:289–300Google Scholar
  4. Boldyrev A, Fedorova T, Stepanova M, Dobrotvorskaya I, Kozlova E, Boldanova N, Bagyeva G, Ivanova-Smolenskaya I, Illarioshkin S (2008) Carnosine [corrected] increases efficiency of DOPA therapy of Parkinson’s disease: a pilot study. Rejuvenation Res 11:821–827.  https://doi.org/10.1089/rej.2008.0716 CrossRefGoogle Scholar
  5. Boldyrev AA, Aldini G, Derave W (2013) Physiology and pathophysiology of carnosine. Physiol Rev 93:1803–1845.  https://doi.org/10.1152/physrev.00039.2012 CrossRefGoogle Scholar
  6. Chaleckis R, Murakami I, Takada J, Kondoh H, Yanagida M (2016) Individual variability in human blood metabolites identifies age-related differences. Proc Natl Acad Sci USA 113:4252–4259.  https://doi.org/10.1073/pnas.1603023113 CrossRefGoogle Scholar
  7. Chengappa KR, Turkin SR, DeSanti S, Bowie CR, Brar JS, Schlicht PJ, Murphy SL, Hetrick ML, Bilder R, Fleet D (2012) A preliminary, randomized, double-blind, placebo-controlled trial of l-carnosine to improve cognition in schizophrenia. Schizophr Res 142:145–152.  https://doi.org/10.1016/j.schres.2012.10.001 CrossRefGoogle Scholar
  8. Chez MG, Buchanan CP, Aimonovitch MC, Becker M, Schaefer K, Black C, Komen J (2002) Double-blind, placebo-controlled study of l-carnosine supplementation in children with autistic spectrum disorders. J Child Neurol 17:833–837CrossRefGoogle Scholar
  9. Csámpai A, Kutlán D, Tóth F, Molnár-Perl I (2004) O-Phthaldialdehyde derivatization of histidine: stoichiometry, stability and reaction mechanism. J Chromatogr A 1031:67–78CrossRefGoogle Scholar
  10. Ditte Z, Ditte P, Labudova M, Simko V, Iuliano F, Zatovicova M, Csaderova L, Pastorekova S, Pastorek J (2014) Carnosine inhibits carbonic anhydrase IX-mediated extracellular acidosis and suppresses growth of HeLa tumor xenografts. BMC Cancer 14:358.  https://doi.org/10.1186/1471-2407-14-358 CrossRefGoogle Scholar
  11. Drozak J, Chrobok L, Poleszak O, Jagielski AK, Derlacz R (2013) Molecular identification of carnosine N-methyltransferase as chicken histamine N-methyltransferase-like protein (hnmt-like). PLoS One 8:e64805.  https://doi.org/10.1371/journal.pone.0064805 CrossRefGoogle Scholar
  12. Gardner MLG, Illingworth KM, Kelleher J, Wood D (1991) Intestinal-absorption of the intact peptide carnosine in man, and comparison with intestinal permeability to lactulose. J Physiol 439:411–422CrossRefGoogle Scholar
  13. Gaunitz F, Oppermann H, Hipkiss AR (2015) Carnosine and cancer. In: Preedy VR (ed) Imidazole dipeptides. The Royal Society of Chemistry, Cambridge, pp 372–392CrossRefGoogle Scholar
  14. Gaunitz F, Hipkiss AR (2012) Carnosine and cancer: a perspective. Amino Acids 43:135–142.  https://doi.org/10.1007/s00726-012-1271-5 CrossRefGoogle Scholar
  15. Gulewitsch W, Amiradzibi S (1900) Ueber das Carnosin, eine neue organische Base des Fleischextraktes. Ber Dtsch Chem Ges 33:1902–1903CrossRefGoogle Scholar
  16. Hipkiss AR, Gaunitz F (2014) Inhibition of tumour cell growth by carnosine: some possible mechanisms. Amino Acids 46:327–337CrossRefGoogle Scholar
  17. Horii Y, Shen J, Fujisaki Y, Yoshida K, Nagai K (2012) Effects of l-carnosine on splenic sympathetic nerve activity and tumor proliferation. Neurosci Lett 510:1–5.  https://doi.org/10.1016/j.neulet.2011.12.058 CrossRefGoogle Scholar
  18. Iovine B, Oliviero G, Garofalo M, Orefice M, Nocella F, Borbone N, Piccialli V, Centore R, Mazzone M, Piccialli G, Bevilacqua MA (2014) The anti-proliferative effect of l-carnosine correlates with a decreased expression of hypoxia inducible factor 1 alpha in human colon cancer cells. PLoS One 9:e96755.  https://doi.org/10.1371/journal.pone.0096755 CrossRefGoogle Scholar
  19. Jackson MC, Kucera CM, Lenney JF (1991) Purification and properties of human serum carnosinase. Clin Chim Acta 196:193–205CrossRefGoogle Scholar
  20. Kaneko K, Smetana-Just U, Matsui M, Young AR, John S, Norval M, Walker SL (2008) cis-Urocanic acid initiates gene transcription in primary human keratinocytes. J Immunol 181:217–224.  https://doi.org/10.4049/jimmunol.181.1.217 CrossRefGoogle Scholar
  21. Lenney JF, George RP, Weiss AM, Kucera CM, Chan PWH, Rinzler GS (1982) Human-serum carnosinase—characterization, distinction from cellular carnosinase, and activation by cadmium. Clin Chim Acta 123:221–231CrossRefGoogle Scholar
  22. Lenney JF, Peppers SC, Kucera-Orallo CM, George RP (1985) Characterization of human tissue carnosinase. Biochem J 228:653–660CrossRefGoogle Scholar
  23. Letzien U, Oppermann H, Meixensberger J, Gaunitz F (2014) The antineoplastic effect of carnosine is accompanied by induction of PDK4 and can be mimicked by l-histidine. Amino Acids.  https://doi.org/10.1007/s00726-014-1664-8 Google Scholar
  24. Nagai K, Suda T (1986) Antineoplastic effects of carnosine and beta-alanine—physiological considerations of its antineoplastic effects. J Physiol Soc Jpn 48:741–747Google Scholar
  25. Okumura N, Takao T (2017) The zinc form of carnosine dipeptidase 2 (CN2) has dipeptidase activity but its substrate specificity is different from that of the manganese form. Biochem Biophys Res Commun 494:484–490.  https://doi.org/10.1016/j.bbrc.2017.10.100 CrossRefGoogle Scholar
  26. Oppermann H, Dietterle J, Purcz K, Morawski M, Eisenlöffel C, Müller W, Meixensberger J, Gaunitz F (2018) Carnosine selectively inhibits migration of IDH-wildtype glioblastoma cells in a co-culture model with fibroblasts. Cancer Cell Int 18:111.  https://doi.org/10.1186/s12935-018-0611-2 CrossRefGoogle Scholar
  27. Ostrom QT, Gittleman H, Xu J, Kromer C, Wolinsky Y, Kruchko C, Barnholtz-Sloan JS (2016) CBTRUS statistical report: primary brain and other central nervous system tumors diagnosed in the United States in 2009–2013. Neuro-Oncology 18:v1–v75.  https://doi.org/10.1093/neuonc/now207 CrossRefGoogle Scholar
  28. Peters V, Jansen EEW, Jakobs C, Riedl E, Janssen B, Yard BA, Wedel J, Hoffmann GF, Zschocke J, Gotthardt D, Fischer C, Köppel H (2011) Anserine inhibits carnosine degradation but in human serum carnosinase (CN1) is not correlated with histidine dipeptide concentration. Clin Chim Acta 412:263–267.  https://doi.org/10.1016/j.cca.2010.10.016 CrossRefGoogle Scholar
  29. Rauen U, Klempt S, de Groot H (2007) Histidine-induced injury to cultured liver cells, effects of histidine derivatives and of iron chelators. Cell Mol Life Sci 64:192–205.  https://doi.org/10.1007/s00018-006-6456-1 CrossRefGoogle Scholar
  30. Renner C, Seyffarth A, de Arriba S, Meixensberger J, Gebhardt R, Gaunitz F (2008) Carnosine inhibits growth of cells isolated from human glioblastoma multiforme. Int J Pept Res Ther 14:127–135.  https://doi.org/10.1007/s10989-007-9121-0 CrossRefGoogle Scholar
  31. Renner C, Zemitzsch N, Fuchs B, Geiger KD, Hermes M, Hengstler J, Gebhardt R, Meixensberger J, Gaunitz F (2010) Carnosine retards tumor growth in vivo in an NIH3T3-HER2/neu mouse model. Mol Cancer 9:2.  https://doi.org/10.1186/1476-4598-9-2 CrossRefGoogle Scholar
  32. Romero SA, Hocker AD, Mangum JE, Luttrell MJ, Turnbull DW, Struck AJ, Ely MR, Sieck DC, Dreyer HC, Halliwill JR (2016) Evidence of a broad histamine footprint on the human exercise transcriptome. J Physiol (Lond) 594:5009–5023.  https://doi.org/10.1113/JP272177 CrossRefGoogle Scholar
  33. Sant M, Minicozzi P, Lagorio S, Børge Johannesen T, Marcos-Gragera R, Francisci S (2012) Survival of European patients with central nervous system tumors. Int J Cancer 131:173–185.  https://doi.org/10.1002/ijc.26335 CrossRefGoogle Scholar
  34. Shen Y, Yang J, Li J, Shi X, Ouyang L, Tian Y, Lu J (2014) Carnosine inhibits the proliferation of human gastric cancer SGC-7901 cells through both of the mitochondrial respiration and glycolysis pathways. PLoS ONE 9:e104632.  https://doi.org/10.1371/journal.pone.0104632 CrossRefGoogle Scholar
  35. Son DO, Satsu H, Kiso Y, Totsuka M, Shimizu M (2008) Inhibitory effect of carnosine on interleukin-8 production in intestinal epithelial cells through translational regulation. Cytokine 42:265–276CrossRefGoogle Scholar
  36. Stupp R, Mason WP, van den Bent MJ, Weller M, Fisher B, Taphoorn MJB, Belanger K, Brandes AA, Marosi C, Bogdahn U, Curschmann J, Janzer RC, Ludwin SK, Gorlia T, Allgeier A, Lacombe D, Cairncross JG, Eisenhauer E, Mirimanoff RO, van Den Weyngaert D, Kaendler S, Krauseneck P, Vinolas N, Villa S, Wurm RE, Maillot MHB, Spagnolli F, Kantor G, Malhaire JP, Renard L, de Witte O, Scandolaro L, Vecht CJ, Maingon P, Lutterbach J, Kobierska A, Bolla M, Souchon R, Mitine C, Tzuk-Shina T, Kuten A, Haferkamp G, de Greve J, Priou F, Menten J, Rutten I, Clavere P, Malmstrom A, Jancar B, Newlands E, Pigott K, Twijnstra A, Chinot O, Reni M, Boiardi A, Fabbro M, Campone M, Bozzino J, Frenay M, Gijtenbeek J, Delattre JY, de Paula U, Hanzen C, Pavanato G, Schraub S, Pfeffer R, Soffietti R, Kortmann RD, Taphoorn M, Torrecilla JL, Grisold W, Huget P, Forsyth P, Fulton D, Kirby S, Wong R, Fenton D, Cairncross G, Whitlock P, Burdette-Radoux S, Gertler S, Saunders S, Laing K, Siddiqui J, Martin LA, Gulavita S, Perry J, Mason W, Thiessen B, Pai H, Alam ZY, Eisenstat D, Mingrone W, Hofer S, Pesce G, Dietrich PY, Thum P, Baumert B, Ryan G (2005) Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:987–996CrossRefGoogle Scholar
  37. Teufel M, Saudek V, Ledig JP, Bernhardt A, Boularand S, Carreau A, Cairns NJ, Carter C, Cowley DJ, Duverger D, Ganzhorn AJ, Guenet C, Heintzelmann B, Laucher V, Sauvage C, Smirnova T (2003) Sequence identification and characterization of human carnosinase and a closely related non-specific dipeptidase. J Biol Chem 278:6521–6531CrossRefGoogle Scholar
  38. Wenig P, Odermatt J (2010) OpenChrom: a cross-platform open source software for the mass spectrometric analysis of chromatographic data. BMC Bioinform 11:405.  https://doi.org/10.1186/1471-2105-11-405 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Klinik Und Poliklinik für NeurochirurgieUniversitätsklinikum Leipzig AöRLeipzigGermany
  2. 2.Institut für Analytische ChemieUniversität LeipzigLeipzigGermany

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