Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1984. / Bibliography: leaves 139-157. / Photocopy. / Microfilm. / xii, 157 leaves, bound ill. 29 cm

Anserine and carnosine are water-soluble dipeptides that have antioxidant properties and are found in the skeletal muscle of mammals and fishes. These dipeptides are removed through the washing process in surimi production. The objective of this research was to determine the concentration of anserine and carnosine in surimi wash water (SWW) at all stages of surimi processing, and undertake preliminary methods to remove and concentrate the two dipeptides and study the effect of surimi wash water extract on color. Wash water samples were collected from a local surimi plant. The samples were treated by the following methods: centrifugaion, heat-treatment at 60, 80 and 100°C and filtration using different ultrafiltration (UF) membranes. The concentrations of the protein and the two dipeptides were analyzed using Lowry and high performance liquid chromatography with a fluorescent detector, respectively. Iron content was determined in SWW samples using atomic absorption spectrometry and colorimetry. Effect of SWW extract and other antioxidants on the color of fresh-farmed salmon were studied using color parameters-hue angle, chroma and lightness. Results showed that there was a trend: content of protein and dipeptides (anserine and carnosine) in SWW (raw) was higher in the first two stages of surimi processing. In the second set of experiment, where different heat treatments were used, it was found that the proteins and dipeptides showed similar trends. Additionally, 80°C followed by 100°C treatment were effective in removal of proteins and recovery of dipeptides. Among UF treatments, 1K molecular weight cut-off membrane was the most effective in recovery of dipeptides. Iron was less than 1 ppm in all SWW samples. Color measurement of fresh farmed salmon patties revealed that treatments of SWW extract (1%) in addition to other food antioxidants such as butylated hydroxy toluene and camosine (1%), mamtained salmon color until day 5. Therefore, SWW extract at lower concentrations may have an economical and potential use as a food antioxidant. / Graduation date: 2000

Thesis (PhD)-Open University. BLDSC no.DX189943.

The antioxidant activity of carnosine and phytic acid was investigated using several model systems. Carnosine and phytic acid alone inhibited metal ion-catalyzed deoxyribose degradation. Carnosine strongly inhibited metal ion-catalyzed lipid peroxidation in liposomes and in ground beef homogenates. Phytic acid facilitated oxidation of Fe (II) to Fe (III), and it inhibited hemeprotein + H202-catalyzed lipid peroxidation in linoleic acid micelles. Antioxidant and color stabilizing effects of carnosine and phytate were investigated in a beef model system. Both compounds increased the rate of pH decline in pre-rigor beef muscle and stabilized fresh meat color by inhibiting metmyoglobin formation and lipid peroxidation in raw samples during storage at 4°C. Both compounds inhibited heme degradation and lipid peroxidation in cooked beef during storage at 4°C. Iron released from heme was strongly related to lipid peroxidation in cooked beef. Ascorbic acid inhibited metmyoglobin formation on the surface of ground beef patties but not in the bulk of the product. In contrast, camosine inhibited metmyoglobin formation and brown color development throughout the product. Carnosine increased cook yield and salt-soluble protein, but ascorbic acid had no effect on cook yield and decreased salt-soluble protein. Carnosine was more effective on inhibition of lipid peroxidation than was ascorbic acid. Phytate greatly enhanced water-holding capacity of raw and cooked meat in a dilute beef model system. Effects of 0.5% sodium phytate, sodium pyrophosphate, and sodium tripolyphosphate, along with 1% NaCl, on physicochemical properties of restructured raw and cooked beef were compared. In raw beef, the treatments with sodium phytate, sodium pyrophosphate, and sodium tripolyphosphate increased meat pH and salt-soluble protein level, and inhibited metmyoglobin formation and lipid peroxidation, compared to the control. In cooked beef, the treatments with sodium phytate, sodium pyrophosphate, and sodium tripolyphosphate increased bind strength, cooked yield, moisture level, and meat pH, and inhibited lipid peroxidation. The treatments with sodium pyrophosphate and sodium tripolyphosphate increased inorganic orthophosphate level in both raw and cooked beef, compared to sodium phytate and the control. These results indicate that carnosine and phytate can be used as meat preservatives for extending shelf-life and enhancing water-holding capacity of meat and meat products.

INTRODUÇÃO: A carnosina é um dipeptídeo com capacidade tamponante presente no músculo esquelético, que pode ser obtido pela ingestão de carnes. Estudos transversais relatam que atletas engajados em exercícios de alta intensidade possuem um maior conteúdo de carnosina muscular (CarnM) comparados a destreinados, sugerindo que o treinamento pode modular a CarnM, apesar da ausência de estudos longitudinais. OBJETIVO: Investigar os efeitos do treinamento intermitente (TI) de alta intensidade sobre a CarnM e seus genes associados. MÉTODOS: Vinte homens saudáveis e vegetarianos (eliminando a influência da dieta) foram pareados pelo consumo máximo de oxigênio (VO2máx), e aleatoriamente designados a um grupo Controle (C, N=10) ou Treinado (T, N=10). O grupo T realizou TI em cicloergômetro 3 dias por semana durante 12 semanas, com progressão do volume (6-12 séries) e intensidade (140-170% do limiar de lactato [LL]). O grupo C manteve a rotina habitual. Antes e após a intervenção, biópsias musculares foram realizadas para a determinação da CarnM, da expressão de genes relacionados à CarnM e da capacidade tamponante muscular in vitro (&#946;&#924;invitro). Foram realizados teste de Wingate e VO2máx para a avaliação do trabalho total (TT), do VO2máx, dos limiares ventilatórios (LV) e do LL. Foi conduzido o Modelo Misto para análise dos dados. RESULTADOS: Um efeito de interação foi observado para CarnM (F = 4.72; P=0.04), com aumentos significantes para o grupo T (Pré: 15.8±5.7 e Pós: 20.6±5.3 mmoL/kg músculo seco; +36.0%, P=0.01) e nenhuma alteração no grupo C (Pré: 14.3±5.3 e Pós: 15.0±4.9 mmoL/Kg músculo seco; +6.3%, P=0.99). Houve melhora no TT, LV, LL, VO2máx e &#946;&#924;invitro no grupo T (todos P<0.05), mas sem mudanças no grupo C (P>0.05). Não houve alteração na expressão gênica das enzimas e transportadores avaliados nos grupos T ou C. CONCLUSÃO: Este estudo sugere que o TI pode aumentar a CarnM, sem alterar os seus genes. Tal aumento, associado ao da &#946;&#924;invitro, pode ajudar a explicar o potente efeito deste tipo de treino sobre a aptidão física e cardiorrespiratória / INTRODUCTION: Carnosine is a dipeptide with buffering capacity present within the skeletal muscle, which can be obtained by meat ingestion. Cross-sectional studies report that athletes engaged in high-intensity exercises have a greater muscle carnosine (MCarn) content compared to their untrained counterparts, suggesting that exercise training can modulate MCarn, despite of the absence of longitudinal studies. OBJECTIVE: To investigate the effects of high-intensity intermittent training (HIIT) on CarnM and its associated genes. METHODS: Twenty healthy and vegetarian men (eliminating dietary influences) were matched by maximal oxygen uptake (VO2máx), and randomly assigned to a Control (C, N = 10) or Trained (T, N = 10) group. The T group performed HIIT on cycle ergometer 3 days per week for 12 weeks, with progressive volume (6-12 series) and intensity (140-170% lactate threshold [LT]). The C group kept their usual routine. Prior to the intervention, muscle biopsies were performed for MCarn determination, expression MCarn-related genes and the muscle buffering capacity in vitro (&#946;&#924;invitro). Wingate and VO2máx tests were performed to evaluate total work done (TWD), VO2máx, ventilatory thresholds (VT) and LT. The Mixed Model was conducted for data analysis. RESULTS: An interaction effect was observed for MCarn (F = 4.72, P = 0.04), with significant increases for the T group (Pre: 15.8 ± 5.7 and Post: 20.6 ± 5.0 mmoL.kg-1 dry muscle; +36%; P = 0.01), but not in C (Pre: 14.3 ± 5.3 and Post: 15.0 ± 4.9 mmoL.kg-1 dry muscle; +6.3%, P = 0.99). There was no change in the gene expression of the enzymes and transporters evaluated in the T or C groups. There was an improvement in TWD, VT, LT, VO2máx and &#946;&#924;invitro in the T group (all P<0.05), but no changes in C (P>0.05). CONCLUSION: This study suggests that HIIT can increase MCarn without altering its genes. This increase, associated with &#946;&#924;invitro, may help to explain the potent effect of this type of training on physical and cardiorespiratory fitness

Das Glioblastom ist mit 70 % aller Gliome der häufigste humane Hirntumor mit sehr ungünstiger Prognose. Es konnte gezeigt werden, dass das natürlich vorkommende Dipeptid Carnosin (β-Alanyl-L-histidin) die Proliferation von Glioblastomzellen inhibiert. Diese Wirkung des Carnosins konnte ebenfalls in vivo nachgewiesen werden. Da Carnosin auch einen Einfluss auf den ATP-Haushalt der Glioblastomzellen besitzt, war das Ziel dieser Arbeit einen Wirkungsort von Carnosin zu identifizieren, womit die ATP mindernden und proliferationshemmenden Eigenschaften erklärt werden können. Es wurde untersucht, ob Carnosin den Energiemetabolismus der Glioblastome beeinflusst. Dabei konnte mithilfe zellbiochemischer Methoden gezeigt werden, dass die untersuchten Zelllinien nicht von der Energieversorgung durch die mitochondriale oxidative Phosphorylierung abhängen, da sich weder Hemmung (KCN) noch Entkopplung (DNP) der Elektronentransportkette auf den zellulären ATP-Gehalt auswirkten. Carnosin hingegen verringerte den ATP-Spiegel dieser Zellen. Die Hemmung der Glykolyse durch Oxamat (LDH-Hemmung), bewirkte einen starken Abfall des intrazellulären ATP-Spiegels, worauf Carnosin keinen zusätzlichen Effekt mehr besaß. Carnosin konnte eine Wirkung auf die glykolytische ATP-Synthese zugesprochen werden. Da ein direkter, molekularer Wirkungsort auf diesem Weg nicht identifiziert werden konnte, wurde parallel untersucht, ob sich über Proteomanalysen der Glioblastomzelllinie T98G ein Wirkungsort, bzw. -mechanismus bestimmen lässt. Anhand der Methode der zweidimensionalen Gelelektrophorese (2D-GE) konnten 31 signifikant differenziell exprimierte Proteine detektiert werden, von denen 6 Proteine (VBP-1, OLA-1, TALDO 1, UROD, BAG-2, GRPEL1) über MALDI-TOF-Analysen identifiziert wurden. In Western-Blot-Analysen konnte ein Protein (VBP-1), neben T98G, auch in primären Glioblastomzelllinien als differenziell exprimiert nachgewiesen werden. Anhand der zellbiologischen und proteinbiochemischen Untersuchungen konnte einerseits eine Verbindung des Carnosins zum HIF1α-Signalweg und andererseits zur generellen posttranslationalen Peptidprozessierung hergestellt werden. Der direkte Nachweis eines Einflusses von Carnosin auf HIF1α wurde aber bisher nicht erbracht.

Das Glioblastoma multiforme (GBM) ist der am häufigsten vorkommende maligne Hirntumor mit äußerst ungünstiger Prognose für die betroffenen Patienten. Typisch für die Tumore ist eine hohe Aktivität der Glykolyse zur Generierung von ATP und zur Bereitstellung von Makromolekülen für die Zellproliferation, während die oxidative Phosphorylierung auch in Gegenwart von Sauerstoff praktisch keine Bedeutung für die Generation von ATP hat, was auch als Warburg Effekt bekannt ist. Das natürlich vorkommende Carnosin (β-Alanyl-LHistidin) wirkt sich antiproliferativ auf Tumorzellen aus, was mit einer Inhibition der glykolytischen ATP Produktion einhergeht. Der Mechanismus der Inhibition ist weitgehend unverstanden und ist Gegenstand der vorliegenden Arbeit. Im Rahmen der durchgeführten Arbeit wurde der Einfluss von Carnosin auf die mRNA Expressionen von für die Glykolyse relevanten Genen untersucht, wobei eine starke Induktion der Pyruvatdehydrogenase Kinase (PDK) 4 in drei GBM Zelllinien beobachtet wurde. Weiterhin konnte gezeigt werden, dass L-Histidin den gleichen Effekt wie Carnosin zeigt, nicht jedoch β-Alanin, L-Alanin oder L-Alanyl-L-Histidin. Da Tumorzellen die intrazelluläre Gewebscarnosinase aber kaum die extrazelluläre Serumcarnosinase exprimieren, liegt die Vermutung nahe, dass die antineoplastische Wirkung des Carnosins auf die enzymatische Spaltung von Carnosin und die daraus resultierende Freisetzung von L-Histidin zurückzuführen ist. In weiteren Untersuchungen wurden Hinweise erbracht, dass Carnosin durch eine Beeinflussung von Histon-Deacetylasen, die endogene PDK4 mRNA Expression steigern könnte. Zusätzlich wurden die Proteinexpressionen der PDK1 und 4 unter dem Einfluss von Carnosin untersucht.

INTRODUÇÃO: A carnosina é um dipeptídeo com capacidade tamponante presente no músculo esquelético, que pode ser obtido pela ingestão de carnes. Estudos transversais relatam que atletas engajados em exercícios de alta intensidade possuem um maior conteúdo de carnosina muscular (CarnM) comparados a destreinados, sugerindo que o treinamento pode modular a CarnM, apesar da ausência de estudos longitudinais. OBJETIVO: Investigar os efeitos do treinamento intermitente (TI) de alta intensidade sobre a CarnM e seus genes associados. MÉTODOS: Vinte homens saudáveis e vegetarianos (eliminando a influência da dieta) foram pareados pelo consumo máximo de oxigênio (VO2máx), e aleatoriamente designados a um grupo Controle (C, N=10) ou Treinado (T, N=10). O grupo T realizou TI em cicloergômetro 3 dias por semana durante 12 semanas, com progressão do volume (6-12 séries) e intensidade (140-170% do limiar de lactato [LL]). O grupo C manteve a rotina habitual. Antes e após a intervenção, biópsias musculares foram realizadas para a determinação da CarnM, da expressão de genes relacionados à CarnM e da capacidade tamponante muscular in vitro (&#946;&#924;invitro). Foram realizados teste de Wingate e VO2máx para a avaliação do trabalho total (TT), do VO2máx, dos limiares ventilatórios (LV) e do LL. Foi conduzido o Modelo Misto para análise dos dados. RESULTADOS: Um efeito de interação foi observado para CarnM (F = 4.72; P=0.04), com aumentos significantes para o grupo T (Pré: 15.8±5.7 e Pós: 20.6±5.3 mmoL/kg músculo seco; +36.0%, P=0.01) e nenhuma alteração no grupo C (Pré: 14.3±5.3 e Pós: 15.0±4.9 mmoL/Kg músculo seco; +6.3%, P=0.99). Houve melhora no TT, LV, LL, VO2máx e &#946;&#924;invitro no grupo T (todos P<0.05), mas sem mudanças no grupo C (P>0.05). Não houve alteração na expressão gênica das enzimas e transportadores avaliados nos grupos T ou C. CONCLUSÃO: Este estudo sugere que o TI pode aumentar a CarnM, sem alterar os seus genes. Tal aumento, associado ao da &#946;&#924;invitro, pode ajudar a explicar o potente efeito deste tipo de treino sobre a aptidão física e cardiorrespiratória / INTRODUCTION: Carnosine is a dipeptide with buffering capacity present within the skeletal muscle, which can be obtained by meat ingestion. Cross-sectional studies report that athletes engaged in high-intensity exercises have a greater muscle carnosine (MCarn) content compared to their untrained counterparts, suggesting that exercise training can modulate MCarn, despite of the absence of longitudinal studies. OBJECTIVE: To investigate the effects of high-intensity intermittent training (HIIT) on CarnM and its associated genes. METHODS: Twenty healthy and vegetarian men (eliminating dietary influences) were matched by maximal oxygen uptake (VO2máx), and randomly assigned to a Control (C, N = 10) or Trained (T, N = 10) group. The T group performed HIIT on cycle ergometer 3 days per week for 12 weeks, with progressive volume (6-12 series) and intensity (140-170% lactate threshold [LT]). The C group kept their usual routine. Prior to the intervention, muscle biopsies were performed for MCarn determination, expression MCarn-related genes and the muscle buffering capacity in vitro (&#946;&#924;invitro). Wingate and VO2máx tests were performed to evaluate total work done (TWD), VO2máx, ventilatory thresholds (VT) and LT. The Mixed Model was conducted for data analysis. RESULTS: An interaction effect was observed for MCarn (F = 4.72, P = 0.04), with significant increases for the T group (Pre: 15.8 ± 5.7 and Post: 20.6 ± 5.0 mmoL.kg-1 dry muscle; +36%; P = 0.01), but not in C (Pre: 14.3 ± 5.3 and Post: 15.0 ± 4.9 mmoL.kg-1 dry muscle; +6.3%, P = 0.99). There was no change in the gene expression of the enzymes and transporters evaluated in the T or C groups. There was an improvement in TWD, VT, LT, VO2máx and &#946;&#924;invitro in the T group (all P<0.05), but no changes in C (P>0.05). CONCLUSION: This study suggests that HIIT can increase MCarn without altering its genes. This increase, associated with &#946;&#924;invitro, may help to explain the potent effect of this type of training on physical and cardiorespiratory fitness

Background Glioblastoma (GBM) is a tumor of the central nervous system. After surgical removal and standard therapy, recurrence of tumors is observed within 6–9 months because of the high migratory behavior and the infiltrative growth of cells. Here, we investigated whether carnosine (β-alanine-l-histidine), which has an inhibitory effect on glioblastoma proliferation, may on the opposite promote invasion as proposed by the so-called “go-or-grow concept”. Methods Cell viability of nine patient derived primary (isocitrate dehydrogenase wildtype; IDH1R132H non mutant) glioblastoma cell cultures and of eleven patient derived fibroblast cultures was determined by measuring ATP in cell lysates and dehydrogenase activity after incubation with 0, 50 or 75 mM carnosine for 48 h. Using the glioblastoma cell line T98G, patient derived glioblastoma cells and fibroblasts, a co-culture model was developed using 12 well plates and cloning rings, placing glioblastoma cells inside and fibroblasts outside the ring. After cultivation in the presence of carnosine, the number of colonies and the size of the tumor cell occupied area were determined. Results In 48 h single cultures of fibroblasts and tumor cells, 50 and 75 mM carnosine reduced ATP in cell lysates and dehydrogenase activity when compared to the corresponding untreated control cells. Co-culture experiments revealed that after 4 week exposure to carnosine the number of T98G tumor cell colonies within the fibroblast layer and the area occupied by tumor cells was reduced with increasing concentrations of carnosine. Although primary cultured tumor cells did not form colonies in the absence of carnosine, they were eliminated from the co-culture by cell death and did not build colonies under the influence of carnosine, whereas fibroblasts survived and were healthy. Conclusions Our results demonstrate that the anti-proliferative effect of carnosine is not accompanied by an induction of cell migration. Instead, the dipeptide is able to prevent colony formation and selectively eliminates tumor cells in a co-culture with fibroblasts.:1 Introduction ........................................................................2 1.1 Glioblastoma ........................................................................................................... 2 1.1.1 Taxonomy, epidemiology and general features of GBM ......................... 2 1.1.2 GBM subtypes and molecular diagnostic .................................................. 3 1.1.3 Therapy ........................................................................................................... 4 1.1.4 GBM cell migration and invasion ................................................................. 5 1.2 Carnosine ................................................................................................................ 6 1.2.1 Chemistry, Biology, Distribution .................................................................. 7 1.2.2 Carnosine homeostasis ................................................................................ 7 1.2.3 Physiological functions.................................................................................. 8 1.2.4 Therapeutic potential ..................................................................................... 9 1.2.5 Carnosine and cancer ................................................................................... 9 1.3 Objective of the study.......................................................................................... 10 2 Publication .......................................................................12 2.1 General information ............................................................................................. 12 2.2 Carnosine selectively inhibits migration of IDH-wildtype glioblastoma cells in a co-culture model with fibroblasts ........................................................................... 13 3 Summary ..........................................................................23 4 References .......................................................................27 5 Appendix ..........................................................................34 5.1 Supplemental material ........................................................................................ 34 5.2 Author’s contribution............................................................................................ 36 5.3 Erklärung über die eigenständige Abfassung der Arbeit ............................... 37 5.4 Curriculum vitae ................................................................................................... 38 5.5 List of publications ............................................................................................... 39 5.6 Acknowledgements ............................................................................................. 41

As one of several imidazole-containing dipeptides, carnosine is found primarily in the skeletal muscle, the brain, the olfactory bulb and the kidneys of mammals, fishes and birds. The enzyme Carnosine Synthase 1 regulates its synthesis and the two enzymes responsible for the dipeptide’s cleavage into its constituent amino acids are known as serum carnosinase (CN1) and tissue carnosinase (CN2). The amino acid L-histidine is supposed to be mainly responsible for the dipeptides physiological properties based on its imidazole moiety. Among the physiological properties ascribed to the dipeptide are its ability to scavenge reactive oxygen species and to protect against advanced glycation end products and lipid peroxidation. Furthermore, the biogenic dipeptide regulates intracellular calcium homeostasis, acts as a pH buffer and as a metal ion chelator. Based on these primary functions, the dipeptide supports mitochondrial activity and diminishes proteotoxicity. Current studies mainly consider these benefits in muscle tissue and refer to cardiovascular and neurodegenerative diseases. In 1986, Nagai and Suda first revealed tumor growth inhibition after using carnosine in a sarcoma mouse model. Later, Holliday and McFarland confirmed these observations in HeLa cells in vitro. Afterwards, Renner et al. demonstrated an anti- proliferative effect of carnosine on human glioblastoma cells. Unfortunately, the dipeptide’s exact molecular mechanisms on tumor cells are still not entirely understood. Another unresolved question is, whether the dipeptide itself is required for the anti- neoplastic effect or whether L-histidine with its imidazole moiety is sufficient and has to be released from carnosine by cleavage of the dipeptide. In order to get a better insight into these questions we investigated the response of glioblastoma cells to L-histidine and carnosine in primary cell cultures and cell lines derived from glioblastoma. Glioblastoma multiforme represents the most common and malignant primary brain tumor. Significant risk factors are still unknown. At diagnosis, the median age is 64 years and the disease is usually found in a progressed stage. Histopathologically, glioblastoma is characterized by necrosis and pronounced mitotic activity in slightly differentiated cells. Accordingly, the tumor shows rapid progression, aggressive invasiveness and, morphological variety. Since 2005, standard of care against glioblastoma follows the STUPP-protocol, which comprises microsurgery, adjuvant chemotherapy with temozolomide and radiotherapy. Nevertheless, it remains one of the most treatment-refractory intracranial tumors; the median over survival after standard treatment is only 14.6 months. Experiments by Letzien et al. demonstrated that L-histidine mimics the anti-neoplastic effect of carnosine in three glioblastoma cell lines investigated. In addition, the amino acid also increased expression of pyruvate dehydrogenase kinase 4 (PDK4) mRNA expression. These observations pointed towards the possibility that carnosine could just be a vehicle, delivering L-histidine to target cells, and that release of the imidazole-containing amino acid is required for the observed effects. In order to investigate whether the effects observed in cell lines are of general significance, cells from ten glioblastoma cell lines and 21 primary glioblastoma cell cultures derived from surgically removed tumors were incubated in a medium containing different concentrations of either carnosine or L-histidine. Cell viability assays measuring the amount of ATP in cell lysates and dehydrogenase activity in living cells were performed. Both substances induced a significant loss of viability. In fact, L- histidine appeared to be even more effective than carnosine, at the same concentration. Next, we investigated whether the enzymes known to be able to cleave carnosine into amino acids are expressed in the cell cultures. Using RT-qPCR, the expression of the mRNA encoding the two enzymes serum carnosinase (CN1, extracellular) and cytosolic or tissue carnosinase (CN2, intracellular) were analyzed in all 31 glioblastoma cell cultures.The experiments revealed high expression of mRNA encoding CN2 in all cultures, whereas expression of CN1 mRNA (gene: CNDP1) was only slightly detectable. Immunoblot performed with ten cell lines revealed that CN2 protein was also present in all cell lines investigated. Therefore, it had to be assumed, that carnosine may be cleaved inside the cells. In a next series of experiments, we investigated whether inhibition of CN2 by the dipeptidase-inhibitor bestatin (ubenimex) does attenuate the effect of carnosine on tumor cell proliferation. Therefore, cell viability was analyzed in the presence of carnosine and in the absence or presence of different concentrations of bestatin. Aside from a general effect of bestatin on cell viability, especially at higher concentrations, no attenuation of carnosine’s antineoplastic effect was observed in the two cell lines investigated. Therefore, we concluded that cleavage of the dipeptide does not seem to be a prerequisite for its effect on tumor cell viability. As we could not rule out that other unknown dipeptidases aside from CN2 may cleave carnosine, we finally measured the intracellular abundances of cells incubated in the absence or presence of carnosine. Therefore, cells from ten cell lines and from five primary cultures were incubated in the absence and presence of either L-histidine or carnosine, and their extracts were subjected to high performance liquid chromatography (HPLC-MS) after derivatization. Although the intracellular abundances of L-histidine of cells incubated in the presence of carnosine clearly demonstrated that the dipeptide is cleaved inside the cells, no correlation between the intracellular amount of L-histidine and the response of cells with regard to viability was observed. Furthermore, the abundance of L-histidine in cells incubated in the presence of 50 mM carnosine was considerably lower, compared to that of cells incubated in the presence of 25 mM L-histidine. As both conditions resulted in a comparable loss of viability, this strongly indicates that cleavage of the dipeptide is not required for its anti-tumor effect and may even be not very efficient. In conclusion, we could confirm that cleavage of carnosine does occur in glioblastoma cells, although this does not raise the intracellular abundance of L-histidine when compared to cells incubated in the presence of the free amino acid. More importantly, cleavage is not required in order to deploy carnosine’s antineoplastic effect. In addition, it appears to be very likely that the imidazole-moiety whether bound or not bound to another amino acid may be sufficient for a therapeutic response. These observations raise a number of interesting questions that should be investigated considering exploiting the antineoplastic effect described for a potential therapeutic use. First of all, the simple question has to be asked, whether it would be sufficient to use L- histidine as an antitumor drug. In that case one has to ask whether sufficient concentrations of L-histidine could be achieved at the side of the tumor when the amino acid is supplemented. Given the fact that it is a proteinogenic amino acid one may suggest, that it is rapidly taken up by other cells. On the other hand, this may also be the case for carnosine. In addition, carnosine is rapidly cleaved by the presence of CN1 in serum. Whether this is in fact a problem is difficult to answer as there are different reports of small clinical trials where carnosine was able to attenuate cognitive impairments after oral supplementation. In addition, the recently identified CN1 inhibitor carnostatine could possibly be supplemented together with carnosine. Another consideration would be to identify other imidazole containing compounds that are no substrate of CN1. However, as it appears that the imidazole-moiety needs to enter the cells the question is, whether other compounds could be transported across the cell membrane. With regard to treatment of brain tumors one should also keep in mind that, aside from the fact, that the blood-brain-barrier is impaired in glioblastoma, it may still be limiting sufficient delivery. At this point, it is also interesting to note that no side effects of carnosine aside from a rarely appearing dysesthesia, are known. However, given the fact that the outcome of current treatment of glioblastoma is still disappointing it appears to be worth to further investigate carnosine’s antineoplastic effect. As the primary targets of the dipeptide are also still widely unknown, the observation that the imidazole moiety is the main effector may help to further elucidate the mechanisms responsible for the antineoplastic effect. At this point it is also interesting to note that the recently discovered benzimidazolinum Gboxin, which also contains an imidazole moiety, exhibits antitumor activity in glioblastoma cells, most likely by irreversibly compromising oxygen consumption. In this case, an elevated proton gradient and a lower pH in cancer cell mitochondria appear to be responsible for the inhibition of oxidative phosphorylation.:1. List of Abbreviations 3 2. Introduction 5 2.1. Glioblastoma 5 Risk factors 5 Localization and histopathology of glioblastoma 5 Molecular pathology 6 Clinic 6 Prognosis and treatment 6 2.2. Carnosine 7 Occurrence 7 Enzymes and transporters 7 Functions 9 Carnosine and cancer 9 Carnosine and its possible application for therapy 10 2.3. Histidine and other histidine-containing compounds 11 L-histidine and naturally occurring dipeptides 11 Physiological functions of L-histidine 11 L-histidine in health and disease 12 L-histidine as a precursor of other metabolites 12 2.4. Objectives of the study 14 3. Publication 15 3.1. General informations 15 3.2. Carnosine’s inhibitory effect on glioblastoma cell growth is independent of its cleavage 16 3.3. Supplemental materials 29 4. Summary 35 5. References 39 6. Appendix 47 6.1. Declaration of independent work 47 6.2. Statement of the own contribution 48 6.3. Acknowledgements 51