Carnosine's Inhibitory Effect on Glioblastoma Cell Growth Is Independent of Its Cleavage

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

Dissertation zur Erlangung des akademischen Grades Dr. med.

an der Medizinischen Fakultät der Universität Leipzig

eingereicht von: Katharina Purcz

Geburtsdatum / Geburtsort: 26.05.1988/Leipzig

angefertigt an der: Universität Leipzig Klinik und Poliklinik für Neurochirurgie

Betreuer: Prof. Dr. Frank Gaunitz Prof. Dr. Jürgen Meixensberger

Beschluss über die Verleihung des Doktorgrads vom: 23.02.2021

1 Inhaltsverzeichnis

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. Curriculum vitae ...... 49 6.4. List of publications ...... 50 6.5. Acknowledgements ...... 51

2 1. List of Abbreviations

AGEs Advanced glycation end products AICAR 5-aminoimidazole-4-carboxamide ribonucleotide ALEs Advanced lipoxidation end products ATP Adenosine triphosphate

BCAN Brevican BMI Body Mass Index

CBTRUS Central Brain Tumor Registry of the United States CD Cluster of Differentiation CN Carnosinase CNS Central Nervous System CO Carbon monoxide COX-2 Cyclooxygenase 2 CS Carnosine Synthase

DLL3 Delta like 3 protein DNA Desoxyribonucleic acid

EC Enzyme Commission EGFR Epidermal Growth Factor Receptor EORTC Organization for Research and Treatment of Cancer

FGFR Fibroblast Growth Factor Receptor

GBM Glioblastoma multiforme GSH Glutathione

HCD Histidine containing dipeptides His Histidine HRG Histidine-rich glycoprotein

IDH Isocitrate dehydrogenase

MET N-methyl-N′-nitroso-guanidine human osteosarcoma transforming gene MG Methylglyoxal MGMT O6-methylguanine DNA methyltransferase mRNA messenger RNA mTORC1 Mechanistic target of rapamycin complex 1

NCIC National Cancer Institute of Canada NF1 Neurofibromin 1 NO Nitric oxide

OS Overall Survival

PCNA Proliferating cell antigen PDGFRA Platelet Derived Growth Factor Receptor Alpha PEPT Oligopeptide transporter 3 PFS Progression-free survival pH Potentia hydrogenii PHT Peptide/histidine transporter PI3 Phosphatidylinositol 3 PI3K Phosphoinositide-3-kinase POT Proton-coupled oligopeptide transporter PRPP 5-phosphoribosyl-1-pyrophosphate PTEN Phosphatase and Tensin homolog qPCR Quantitative Real Time Polymerase Chain Reaction

RNA Ribonucleic acid RNS Reactive nitrogen species ROS Reactive oxygen species RT Radiotherapy RTK Receptor tyrosine kinase RT-qPCR Reverse Transcription qPCR

STAT3 Signal transducer and activator of transcription 3

TMZ Temozolomide TTF Tumor treating fields

VEGF Vascular endothelial growth factor

WHO World Health Organization

YKL40 also known as Chitinase-3-like protein 1 (CHI3L1)

4 2. Introduction

2.1. Glioblastoma

Appearing with a large clinical and histopathological heterogeneity, glioblastoma (GBM) represents the most common and malignant brain tumor. The WHO classifies glioblastoma as a grade IV tumor, which is estimated to occur with an annual incidence of 3.20 per 100 000 population in the US. With a percentage of 55.4% it takes up the majority of primary and other CNS gliomas [98]. This oncologic disease is most common at a median age of 64 at the moment of diagnosis and has its highest incidence in patients aged 65 years and older [84, 98]. In addition, its occurrence rate is almost 1.57-times higher among men than women, and it appears 1.93-times more frequently in Whites than in Afro-Americans [98].

Risk factors

There is a huge list of factors, suspected of causing malignant brain tumors: vinyl chloride, pesticides, smoking, petroleum refining, synthetic rubber manufacturing, as well as electromagnetic fields, formaldehyde or nonionizing radiation from cell phones [27]. However, none of the previous clinical trials could ensure significant risk factors [87]. Solely evidence for ionizing radiation has been confirmed to cause GBM tumor development. Furthermore, hereditary diseases such as neurofibromatosis 1 and 2, tuberous sclerosis, Li-Fraumeni syndrome, retinoblastoma or Turcot syndrome are linked to a higher risk of developing high and low grade brain tumors [27], but only 5% of glioma patients exhibit a positive familial anamnesis [87].

Localization and histopathology of glioblastoma

With a frequency of 61%, primary gliomas appear in one of the four brain lobes: 25% frontal lobe, 20% temporal lobe, 13% parietal lobe and 3% occipital lobe [27]. Uncommon in the spinal cord and cerebellum, glioblastomas often emerge in the brainstem of children, known as malignant brainstem tumor glioma. Spreading through the corpus callosum and arising with symmetric and bilateral extension, the "butterfly glioma" depicts an often observed manifestation [66]. Whereas apoptotic cell demise is rare, the most distinctive feature of glioblastoma is the occurrence of necrosis (~80% of the total tumor mass) caused by increased tumor cell proliferation resulting in insufficient perfusion. Hypoxia on the other hand causes an intensive excrescence of blood vessels, allowing aggressive growth and promotion of tumor invasivity. By contrast, vascular penetration and consequently hematogenous dissemination, as well as extraneural metastasis, remain rare [66]. Histopathologically, a pronounced mitotic activity in slightly differentiated cells completes the pleomorphic cellular structure [66]. Moreover, small, but also possibly giant cells, partially hyperchromatic nuclei with atypia generate the microscopic image of the WHO IV graded tumor [81].

5 Typically, all gliomas show a tendency to progress towards a less differentiated, but more malignant phenotype [66].

Molecular pathology

Currently, molecular and genetic analyses are gaining increased importance for the classification and the prognosis of glioblastomas. The determination of isocitrate-dehydrogenase 1 (IDH) enables a distinction between primary and secondary GBM. IDH-wildtype is found in de novo appearing primary tumors (~90% of glioblastomas), and secondary GBMs, arising from precedent lower grade gliomas, are characterized by a mutated version (~10% of glioblastomas). The median age at diagnosis is ~62 years for patients presenting with a primary GBM, whereas the median age at diagnosis of secondary GBM is ~44 years. IDH-mutant GBM is also characterized by a better prognosis: after surgery, chemotherapy and radiotherapy, the median overall survival is around 31 months compared to 15 months for primary GBM [82]. With regard to standard therapy consisting of radiotherapy and adjuvant chemotherapy with temozolomide (TMZ), the O⁶-methylguanine–DNA methyltransferase (MGMT) became a valuable marker for prognosis. MGMT is a DNA-repair enzyme, which attenuates the effect of the alkylating chemotherapeutic TMZ by removing alkyl groups. Some gliomas reveal an epigenetically silenced modification in the MGMT promoter and are therefore more susceptible to chemotherapy and even show better response to ionizing radiation, leading to better survival rates [49]. 67.3% of GBMs are associated with no less than one mutation in receptor tyrosine kinases (RTK), which are essential regulators of growth factor signaling pathways, regulating proliferation, metabolism and thus survival of cells [39]. Exemplarily, mutations in the Epidermal Growth Factor Receptor (EGFR) are found in 57.4%, in the Platelet Derived Growth Factor Receptor Alpha (PDGFRA) in13.1%, in the Fibroblast Growth Factor Receptor (FGFR) in 3.2% and in the N-methyl-N′-nitroso-guanidine human osteosarcoma transforming gene (MET) in 1.6% of GBMs. Nearly one of four GBMs also shows alterations in phosphatidylinositol (PI3) kinase signaling [22].

Clinic

A remarkably short clinical history, less than three months without treatment, is distinctive of GBM patients [66]. Depending on tumor localization and growth rate, patients present with symptoms of increased intracranial pressure, comprising nausea, headache, vomiting and personality changes. Suspected clinical symptoms for an intracranial space-consuming process are focal-neurologic failures and cognitive deficits. Emerging focal or general epileptic seizures occur in one out of four GBM patients up to about one in two over the course of tumor progression [27, 42]. At an advanced stage, patients additionally suffer from drowsiness, dysphagia, incontinence and from common end-of-life symptoms, such as fatigue, anorexia and pain [109].

Prognosis and treatment

The current standard of care for GBM is initial surgical resection followed by treatment with a combination of the alkylating agent TMZ and radiotherapy (RT). 6 Besides their morphological variety, GBMs are typically characterized by miserable prognosis and bleak quality of life [89]. According to the Central Brain Tumor Registry of the United States (CBTRUS), a one-year-survival-rate of 39.3% and a five-year-survival- rate of 5.5% are documented [98]. In reference to the European Organization for Research and Treatment of Cancer (EORTC) and the National Cancer Institute of Canada (NCIC), the 5-year overall survival (OS) of patients who received RT and TMZ is five-fold higher than of patients who received just RT (9.8% vs. 1.9%) [84]. Owing to the STUPP-protocol, which combines a therapy of ionizing radiation and temozolomide after surgery, the current median OS has improved to 14.6 months. This implies a survival gain of 2.5 months, as compared to radiotherapy alone [115]. Former studies, considering a time scale from 2000-2003, documented a median OS of 8.1 months [106]. Since the approval for tumor treating fields (TTF) in October 2015, a new potential manner of treating GBM locoregionally is obtainable. The so called Optune® intends to interrupt the cell cycle during metaphase, leading to apoptosis and cell death. By delivering low-intensity, intermediate-frequency alternating electric fields via transducer arrays, applied to the patient’s shaved scalp, the TTFs alone achieve an ameliorated quality of life, but neither a longer OS nor progression-free survival (PFS) [114]. However, compared to TMZ therapy alone, the combination of Optune® with TMZ ameliorates the PFS from 4.0 months up to 7.2 months and the median OS time from 16.6 to 19.6 months, without any increased systemic side effects [27, 114] .

2.2. Carnosine

During chemical analyses of Liebig’s extract of meat, Russian scholars of the University of Charkow, Gulewitsch and Amiradzibi, isolated the hitherto unknown dipeptide L- carnosine (β-alanyl-L-histidine) in 1900 [43]. The synthetic enantiomorph D-carnosine is the chemical compound of D-histidine and, again, ß-alanine [124]. The present work refers to the endogenous L-enantiomer L- carnosine.

Occurrence

In fishes, birds, and mammals, the greatest amounts of carnosine were found in skeletal muscle, brain, and kidney tissue [64]. Up to 100 mmol/kg in dry muscle mass were measured [32]. In mammalian tissue, its occurrence is most notably restricted to skeletal muscle and the olfactory bulb. Nevertheless, small quantities also occur in other juices and parts of the mammalian organism, such as neurons, glial- and hypothalamic cells and other certain brain regions [21, 64, 83]. Interspecies variations have been observed [117].

Enzymes and transporters

The two amino acids β-alanine and L-histidine make up the structure of the naturally occurring L-carnosine [124]. Characterized by no strict substrate specificity, the linking enzyme is called carnosine synthase (CS; EC 6.3.2.11) and is part of the ATP-grasp family of ligases [18, 31, 63]. Recently reported investigations disclose the existence of a 7 second enzyme, the imidazole dipeptide synthase. In contrast to carnosine synthase, no ATP is required for its activity [18, 121]. Whereas its compound L-histidine seems to be responsible for most features of the dipeptide, ß-alanine is known as the principal regulator of its synthesis [18]. In several fishes, histidine containing dipeptides (HCD) are replaced by free L-histidine. The imidazole ring of L-histidine is suggested to be the main agent for the typical characteristics of HCD, as specified below [1, 2, 17, 18, 26]. In particular, the pH- buffering activity is attributed to the imidazole ring, more precise its nitrogen atoms [18, 120]. First described in 1949 in swine kidney [47], two types of carnosinases undertake the role to hydrolyze the dipeptide into its two amino acids. Carnosinase 1 (CN1; EC 3.4.13.18) is also called serum carnosinase. Carnosinase 2 (CN2; EC 3.4.13.20), on the other hand, acts in the cytosol and is also named tissue carnosinase. The associated genes CNDP1 and CNDP2 are localized on chromosome 18 [74]. The level of CN1 depends on age and sex: almost quiescent after birth, its activity increases in adults [118]. Females exhibit higher expression of the specific dipeptidase than their male peers [34]. Whereas CN1 is only represented in human brain, serum, liver, and kidney glomeruli, CN2 shows a widespread distribution, albeit excluding serum and cerebrospinal fluid (CSF) [75, 76, 118]. Previous studies summarize the typical histological expression of regulating enzyme groups as follows: A pronounced dominance either of synthesizing enzymes (CS) or of cleaving enzymes (CN1 and CN2), characterizes different tissue subtypes. These observations additionally exclude the combination of both enzyme forms in one cell type. As appropriate, CS prevails in muscular cells [48]. Hereby, the intramuscular concentration of the HCD depends on human age: It is low in children and increases with age up to adolescence, remains constant in adulthood, and declines in senescence [6]. In contrast to the serum-specific isoform, CN2 can be inhibited by the statin ubenimex, which is sold under the trademark bestatin [75]. Bestatin is produced by the actinomycete Streptomyces olivoreticuli and has been established as [(2S, 3R)-3-amino- 2-hydroxy-4-phenylbutanoyl]-L-leucine [116]. Initially discovered in 1976, Umezawa et al. showed bestatin to be a reversible competitive inhibitor of aminopeptidase B (EC 3.4.11.6) and leucine aminopeptidase (EC 3.4.11.1) [122, 123]. It further inhibits Zn²⁺- binding aminopeptidases, mainly the aminopeptidase N (equal to CD 13/EC 3.4.11.2). In mice with diabetic retinopathy, Hossain et al. recently showed significant retardation of retinal vascular permeability and leukostasis after intravitreal injection of bestatin in its role as a CD13 inhibitor [55]. Known as an immunomodulatory agent, bestatin is available as an anti-cancer drug in Japan. There, it has been approved as a supplementary drug, notably to the chemotherapeutic treatment of non-lymphatic leukemia. Several works showed induced apoptosis and division arrest in normal and tumor cells treated with bestatin. Recently, promising results in the therapeutic approaches of acute myeloid leukemia, lung squamous cell carcinoma and other cancer types have been reported [96]. In addition, bestatin can improve inflammatory processes, due to its contribution to the production of cytokines and chemokines triggered by macrophages and monocytes [79]. Up to now, neither antimycotic, antibacterial nor deeply toxic effects have been secured [123]. Four different transporters of the proton-coupled oligopeptide transporter family (POT- family) that facilitate the passage of substances through the cellular membrane are

8 described: Oligopeptide transporter 1 and 2 (PEPT1 and 2), as well as peptide/histidine transporter 1 and 2 (PHT1 and 2). PEPT1 is primarily situated in the small intestine and is supposed to be a low-affinity transporter of high-capacity. In contrast, PEPT2 reabsorbs filtered peptides in the renal tubules and is denoted by high-affinity and low- capacity [18]. These transporters act in further (human) tissues. Exemplarily, PEPT2 was notably found in the choroid plexus, astrocytes, and in epithelial cells [12, 18, 111]. So far, only little is known about the PHT transporters. Although PHT1 is found in skeletal muscles and PHT2 is known to be expressed in the placenta, lungs, heart, leukocytes, and spleen, the transport direction across the cellular membrane and its precise functions presently remain unclear [18]. Recently, Oppermann et al. published 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 [91].

Functions

Freely occurring in myoplasm, the histidyl dipeptide plays a pivotal role in skeletal muscles: Fast-twitch, white muscle cells contain more carnosine than the red muscle- fiber type, indicating advantages for anaerobic muscular efforts. Hereby, the peptide is considered to intervene in lactic acidosis as a mobile pH-buffering agent [29]. Accruing protons can be quenched and, equally, reactive oxygen species (ROS) scavenged. In addition, the release of intramuscular calcium and its sensitivity are promoted [11]. Due to its cleaving enzyme, carnosinase, the interstitial histidine and histamine availability are accordingly advanced [41]. In contracting muscles, therefore, supplemental carnosine intake can contribute to homeostasis [7, 18, 19]. Further potential roles, such as stimulation of myosin ATPase, and influence on glycolytic processes, on oxidative phosphorylation and on neuromuscular improvement remain topic of current investigations [16]. Thus, a wide range of beneficial functions for the human being can be attributed to the biogenic dipeptide. Decreasing ROS [14], as well as reactive nitrogen species (RNS) [36], acting as a metal ion chelator and hydrogen ion buffer, and as ameliorating metabolism, are only a few of them. These qualities are conditioned by decreasing glycolysis, supporting mitochondrial activity, and diminishing proteotoxicity [51]. In like manner, influences on gene expression, protein phosphorylation and mRNA translation were referred to carnosine [56, 72, 112]. Zinc and copper are the most important metal ions, characterized to form effective physiological complexes with the bioactive peptide [8]. Exemplarily, as a chelator of zinc, the complex (pharmaceutically also known as polaprezinc) is able to protect stomach mucosa against peptic ulcers and has been detected as a potential compound against Helicobacter pylori-associated gastritis [45, 60, 67].

Carnosine and cancer

In 1986, Nagai and Suda first revealed tumor growth regression and reduced mortality in vivo after carnosine application in a sarcoma mouse model [86]. Ten years later, Holliday and McFarland confirmed carnosine’s anti-neoplastic activity in vitro in HeLa cells.

9 In previous years, several investigations were performed at the Department of Neurosurgery at the University of Leipzig examining carnosine’s effects on glioblastoma. In 2008, Renner et al. demonstrated that the dipeptide reduced viability of cultured glioblastoma cells isolated from surgically removed tumors [100]. Later, the same group investigated the peptides’ influence in an NIH3T3-HER2/neu mouse model [101] demonstrating decreased tumor cell proliferation and lower mitotic rates under the influence of carnosine also in vivo. However, the exact mechanisms responsible for carnosine’s antineoplastic effect are still unknown and its primary targets have not unequivocally been identified. Some experiments point towards the possibility that the dipeptide affects glycolytic ATP production and may also have an effect on oxidative phosphorylation [92, 99]. In 2014, Letzien et al. demonstrated that carnosine increases the expression of pyruvate dehydrogenase 4 (PDK4) mRNA in glioblastoma cells without affecting the enzymatic activity of PDK4 [77], and recently the effect of carnosine on PDK4 mRNA expression was shown to be dependent on the induction of histone acetylation [90]. However, neither a primary histone deacetylase (HDAC) nor a histone acetylase (HAT) targeted by carnosine was identified. Experiments performed by other groups who investigated the antineoplastic activity in different tumor models also suggested that the dipeptide may affect signal transduction. Investigating carnosine’s effect on Caco-2 (colorectal adenocarcinoma cells), Fujii et al. reported that carnosine may act through activation of cAMP responsive element binding protein (CREB)-related pathways by activating Ca2+-related pathways [38]. In addition there are hints that in HCT116 colon carcinoma cells an influence on ERK1/2 phosphorylation [58] or HIF-1 alpha signaling may play pivotal roles [59]. However, whereas Forsberg et al. [37] and Iovine et al. [59] reported inhibition of HIF-1 alpha signaling, Ditte et al. reported its increase to be associated with growth inhibition [30]. More recently, Lee et al. also demonstrated that in HCT 116 cells carnosine suppresses NF-κB/STAT1 signaling, inducing cell cycle arrest and apoptosis [73].

Carnosine and its possible application for therapy

The central issue, considering using carnosine as an orally given drug, is its fast degradation by serum carnosinase, CN1 [50]. However, there are several reports in the literature, describing trials, in which carnosine has successfully been used for the treatment of diseases. As reported by Chez et al., statistically significant improvements were observed in children with autistic spectrum disorders receiving a daily dose of 800 mg carnosine per os for 8 weeks [25]. Comparable benefits, due to oral carnosine therapy, were reported in patients with schizophrenic symptoms [24], and in Persian Gulf War veterans, suffering from cognitive impairment [9]. In addition, Boldyrev et al. [15] demonstrated, that a concomitant therapy of L-DOPA and carnosine amended Parkinson-related neurological disorders. Therefore, it appears to be likely, that the orally received carnosine may be protected from serum carnosinase or may be resynthesized at the target side. Recently, Qiu et al. discovered carnostatine (SAN 9812), a potent and selective inhibitor of the serum carnosinase. Following subcutaneous injection of the inhibitor in a mouse model, especially in addition with carnosine treatment, lower CN1 levels were ascertained.

10 Moreover, the authors describe a positive effect on type 2 diabetes with diabetic nephropathy, typically characterized of high CN1 serum levels. In this case, new found carnostatine could be a protective agent against metabolic complications, such as diabetic nephropathy, in particular as long term treatment [97].

2.3. Histidine and other histidine-containing compounds

In their investigation on carnosine’s effect on PDK4 mRNA expression, Letzien et al. could demonstrate that a comparable effect on expression was observed when the cells received L-histidine [77]. In addition, the authors could demonstrate that the amino acid significantly inhibited tumor cell growth. As the main goal of the work presented was to evaluate whether L-histidine needs to be cleaved in order to release this moiety, a more detailed introduction about the physiological functions of L-histidine and other imidazole containing dipeptides is given in the following sections.

L-histidine and naturally occurring dipeptides

In 1896, Albrecht Kossel and Sven G. Hedin succeeded in isolating, independently of each other, the basic amino acid histidine from the sperm of a sturgeon and from casein, respectively [46, 62, 70]. The name histidine is derived from the Greek word "istos" (ίστός = tissue) [46]. Whereas D-histidine is of minor significance, the L-enantiomer is the naturally occurring form [108] and a proteinogenic amino acid. After the isolation of carnosine, identifying the first naturally occurring dipeptide being composed of L-histidine and another amino acid in 1900 [43], a methylated derivative, designated anserine (ß-alanyl-1-N-methylhistidine), was discovered in 1929 by Tolkachevskaya et al. in chicken [119] and later by Ackermann et al. in goose muscles [3]. Shortly afterwards, ophidine (balenine; ß-alanyl-3-N-methylhistidine) [28], was found in snakes and whales [18, 20, 126].

Physiological functions of L-histidine

Histidine is known to be crucial for the production of erythrocytes. As part of the α- and ß-chains [68], histidine makes up 8% of hemoglobin [57]. Being described as proximal His F8 (8th amino acid, helix F) and distal His E7 (7th amino acid, helix E), histidine prevents oxidation of the iron and lowers its CO affinity. In this way, histidine contributes to the protection against carbon monoxide, which arises from porphyrin degradation [93]. Histidine is the main amino acid responsible for the Bohr Effect, which is caused by buffering protons in the blood. This effect refers to the decreasing affinity of hemoglobin to oxygen that occurs when pH declines or carbon dioxide accumulates [94]. Analogous to hemoglobin, myoglobin also contains His F8 and His E7 helices in its α- and ß-subunits, to enable the storage and transport of oxygen in the muscle cells of mammalians [113]. Furthermore, histidine forms the largest part of the histidine-rich glycoprotein (HRG). This single polypeptide chain is synthesized in hepatocytes and is mainly found in the plasma of vertebrates. Because of its ability to regulate several important biological 11 processes, the glycoprotein is gaining importance. As a possible heparin-antidote, HRG inhibits the interaction between heparin and antithrombin 3. Antiangiogenic effects and a decrease in tumor size have been demonstrated in a fibrosarcoma-bearing mouse model. In addition, a high affinity of the HRG, mainly to divalent ligands, such as Zn2+, tropomyosin, heme, plasminogen, plasmin, fibrinogen, thrombospondin, and heparin, has been found and especially ascribed to the histidine-rich region of the HRG, depending on pH and histidine residues [61].

L-histidine in health and disease

Inflammatory responses, malnutrition, oxidative stress, and a significantly higher mortality were observed in people suffering from chronic kidney disease associated with low plasma levels of histidine. Hereby, the supplementation of L-histidine in chronic renal failure patients leads to fewer complications, such as anemia, inflammation, and oxidative stress. Rather, it enhances the patients' general nutritional condition and their nitrogen balance [125]. Kopple and Swendseid found evidence for histidine being an essential amino acid in healthy adults and in people suffering from kidney diseases: by means of a histidine- deficient diet, they observed typical skin lesions, impaired erythropoiesis, declining reticulocytes, and elevated iron levels in the blood. Also, nonspecific effects, such as anorexia, discomfort, fatigue, and behavioral modifications, were noted. After supplementing L-histidine, the symptoms regressed, the hematopoiesis normalized, and the nitrogen metabolism improved [69]. Current studies demonstrate regression of cataracts in salmon lens, following dietary L- histidine supplementation [103]. Several investigations demonstrated that patients suffering from rheumatoid arthritis had significantly lower levels of free serum histidine compared to healthy individuals [40, 65]. However, therapeutic L-histidine supplementation revealed no benefit in rheumatic patients [95]. Feng et al. examined obese women with metabolic syndrome after histidine therapy, ascertaining improved insulin resistance, lower BMI and body fat percentage, as well as repressed levels of pro-inflammatory cytokines and oxidative stress after 12 weeks of histidine substitution. At the same time, neither side effects nor abnormal serum parameters could be detected [35].

L-histidine as a precursor of other metabolites

By means of histidase (E.C.4.3.1.3, also called histidine ammonia-lyase or histidinase), ammonium is eliminated [105]. Thus, L-histidine is converted to urocanate, which is cleaved to 4(5)-imidazolone-5(4)-propionic acid by urocanase. As a precursor of L- glutamate, the intermediate N-formino-L-glutamate is enzymatically formed by formininoglutamase (Acetyltransferase). Consequently, L-histidine belongs to the glucogenic amino acids [23]. Furthermore, L- histidine can be metabolized by the enzyme histidine decarboxylase (E.C.4.1.1.22) [10]. Under the elimination of carbon dioxide, histamine (4- imidoazolethylamine or 4-(2’-aminoethyl)imidazole [110]) accrues [33]. This biogenic amine, e.g. produced in basophile granulocytes, mast cells, platelets, in the stomach,

12 smooth muscle cells and in the brain, is stored inside the granules of mast cells. Histamine is responsible for vasodilatation, vascular permeability, gastric acid production or smooth muscle contractions [44, 78, 85, 102]. Interestingly, L-histamine can promote or antagonize cancer progression. By activating histamine H₂-receptors on the surface of host T-suppressor lymphocytes, histamine leads to inhibited cell-mediated cytotoxicity. Thereby, H₂-receptor antagonists may counteract the immunosuppression. However, histamine also stimulates histamine H₁- receptors on T-effector cells. Hence, H₁-receptor inhibitors would repress the immunostimulatory influence of histamine [10]. Several studies have investigated the anti-neoplastic effects of cimetidine, a substituted imidazole compound, acting as a H₂- receptor antagonist. Cimetidine is commonly prescribed to treat or prevent gastric ulcers. In some cancers, the H₂-inhibitor also possesses immunomodulating and anti- angiogenetic properties and impedes tumor cell cohesion. Consequently, it influences the tumor micro environment and is supposed to abolish tumor cell growth [71]. Adam et al. [4] showed significantly induced apoptosis in human colorectal cancer cell lines after treatment with cimetidine. Nevertheless, the opinions concerning cancer development in association to antihistamine exposition are divergent. Scheurer et al. [104] found a higher tendency of anaplastic glioma in patients with long-term intake of antihistamines, as compared to controls.

13 2.4. Objectives of the study

Fighting for a better prognosis for patients with glioblastoma multiforme still remains a challenge, even for patients who received surgery and adjuvant radio-chemotherapy [115]. Despite decades of research, the rapid progression, the infiltrative nature, and the localization of the most common primary brain tumor still determine a lack of potent therapy targets, resulting in its grim prognosis [84, 98]. The dipeptide carnosine (ß-alanyl-L-histidine) had been shown to inhibit tumor cell growth [54, 86, 101]. As its component L-histidine mimics this effect [77], the present study investigated whether cleavage of carnosine and release of L-histidine is required for its anti-neoplastic effect in order to get a better insight into the role of L-histidine and pharmacological issues related to using carnosine or potential derivatives of it for therapy. In order to verify that L-histidine mimics the effect of carnosine on tumor cell growth and PDK4 mRNA expression, cells from 10 glioblastoma cell lines and 21 cell cultures derived from glioblastoma patients were investigated with regard to viability and expression of PDK4 mRNA under the influence of both compounds. In addition, the expression of the carnosine-cleaving enzymes CN1 and CN2 was investigated, employing RT-qPCR and immunoblots. Furthermore, the dipeptidase-inhibitor bestatin was used to investigate the response of cells to treatment with carnosine when carnosinase 2 is inhibited, to reveal whether cleavage of the dipeptide is required for its anti-neoplastic effect. Using HPLC-MS, the intracellular availability of carnosine and L-histidine was determined after treatment of cells with carnosine or L-histidine.

14 3. Publication

3.1. General informations

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

Authors: Oppermann, Henry *; Purcz, Katharina *; Birkemeyer, Claudia; Baran-Schmidt, Rainer; Meixensberger, Jürgen; Gaunitz, Frank *: these authors contributed equally

Journal: Amino Acids

Impact factor: 2.52

Volume: 51

Issue: 5

Pages: 761 – 772

Received: 5 November 2018

Accepted: 18 Februar 2019

Published: 12 March 2019

References: 38

Language: English

Publishing house: BioMed Central Ltd, Springer GmbH

15 3.2. Carnosine’s i nhi bit ory effect on gli oblastoma cell growth is independent of its cleavage

16 Amino Acids (2019) 51:761–772 https://doi.org/10.1007/s00726-019-02713-6

ORIGINAL ARTICLE

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

Henry Oppermann1 · Katharina Purcz1 · Claudia Birkemeyer2 · Rainer Baran‑Schmidt1 · Jürgen Meixensberger1 · 1 Frank Gaunitz

Received: 5 November 2018 / Accepted: 18 February 2019 / Published online: 12 March 2019 © Springer-Verlag GmbH Austria, part of Springer Nature 2019

Abstract The naturally occurring dipeptide carnosine (β-alanyl-L-histidine) inhibits the growth of tumor cells. As its component L-histidine mimics the eff we investigated whether cleavage of carnosine is required for its antineoplastic eff Using ten glioblastoma cell lines and cell cultures derived from 21 patients suff ing from this malignant brain tumor, we determined cell viability under the infl 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 eff We observed a signifi y 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 signifi release of L-histidine in cells incubated in the presence of 50 mM carnosine (p < 0.05), which did not correlate with carnosine’s eff on viability. Furthermore, the carnosinase 2 inhibitor bestatin did not attenuate carnosine’s eff 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 infl by L-histidine and carnosine, but did not correlate with eff on viability. In conclusion, we demonstrate that the antineoplastic eff of carnosine is independent of its cleavage.

Keywords Carnosine · L-Histidine · Glioblastoma · Carnosinase · Pyruvate dehydrogenase kinase 4

Introduction

In 1900, Gulewitsch and Amiradzibi investigated the Handling Editor: W. Derave. chemical components of Liebig’s meat extract (Gul- ewitsch and Amiradzibi 1900), discovering the first Henry Oppermann and Katharina Purcz equally contributed. peptide, carnosine (β-alanyl-L-histidine). Carnosine is one of several imidazole-containing dipeptides such as Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00726-019-02713-6) contains homocarnosine (γ-aminobutyryl-L-histidine) and anserine supplementary material, which is available to authorized users.

* Henry Oppermann Frank Gaunitz [email protected] [email protected]

Katharina Purcz 1 Klinik Und Poliklinik für Neurochirurgie, [email protected] Universitätsklinikum Leipzig AöR, Forschungslabore, Claudia Birkemeyer Liebigstraße 19, 04103 Leipzig, Germany [email protected] 2 Institut für Analytische Chemie, Universität Leipzig, Rainer Baran-Schmidt 04103 Leipzig, Germany [email protected] Jürgen Meixensberger [email protected]

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(β-alanyl-N-π-methyl-L-histidine), which are present in high intact molecule or one of its moieties (β-alanine/L-histidine) concentrations in the central nervous system and the skeletal is responsible for tumor growth inhibition. Previously, we muscle of vertebrates (Boldyrev et al. 2013). In the human reported that L-histidine is able to mimic the antineoplas- body, carnosine levels are controlled by three enzymes. The tic eff of carnosine in glioblastoma cell lines (Letzien formation of imidazole-containing dipeptides is catalyzed et al. 2014). In these experiments, L-histidine also induced by the ATP-requiring Carnosine Synthase 1 [EC 6.3.2.11, expression of pyruvate dehydrogenase kinase 4 (PDK4) also known as ATP-Grasp Domain-Containing Protein 1 in a comparable manner to carnosine. L-histidine released (ATPGD1)]. The enzyme is encoded by the CARNS1 gene, from the dipeptide may then become a substrate for further which is in humans primarily expressed in skeletal muscle, reactions such as decarboxylation or deamination leading to heart muscle, and olfactory neurons (Drozak et al. 2013). histamine or urocanate formation, respectively. In fact, both The degradation of carnosine can be carried out by two compounds are known to be able to induce gene expression metalloproteases. The extracellular occurring carnosine (Romero et al. 2016; Kaneko et al. 2008). Therefore, we dipeptidase 1 (CN1; EC 3.4.13.20; also known as serum hypothesized that cleavage of the dipeptide may be required carnosinase and encoded by the CNDP1 gene) is supposed to to deploy its eff be mainly produced in brain (Jackson et al. 1991), although it may also be synthesized in the liver (Peters et al. 2011). It hydrolyses carnosine with high specifi , but is also able Materials and methods to cleave anserine (β-alanyl-3-methyl-L-histidine), L-alanyl- L-histidine, L-glycyl-L-histidine, and homocarnosine (deam- Reagents ino-3-aminomethyl-alanyl-L-histidine) (reviewed in Bellia et al. 2014). The second enzyme (CN2; EC 3.4.13.18; also Unless stated, otherwise, all chemicals were purchased known as cytosolic non-specifi dipeptidase or formerly from Sigma-Aldrich (Taufkirchen, Germany). Carnosine known as tissue carnosinase; encoded by the CNDP2 gene), was kindly provided by Flamma (Flamma s.p.a. Chignolo is expressed intracellularly and occurs ubiquitously in human d’Isola, Italy). tissue (Teufel et al. 2003). Although CN2 was shown to have optimum activity at pH 9.5 with regard to cleavage of carno- Cell lines and primary cell cultures sine (Lenney et al. 1985), the dipeptide can be hydrolyzed intracellularly, but at a much lower rate compared to other The glioblastoma cell lines G55T2, 1321N1, and U373 were histidine containing dipeptides, as demonstrated by experi- obtained from Sigma-Aldrich (Taufkirchen, Germany), the ments with HEK293T cells (Okumura and Takao 2017). cell lines U87, T98G, and LN229 from the American-Type Since the discovery of carnosine, several physiological func- Culture Collection (ATCC; Manassas, USA); MZ54 and tions have been ascribed to it, such as Ca2+ regulation, pH- MZ18 were originally obtained from Donat Kögel (Frank- buff ing, metal ion chelating, scavenging of reactive oxygen furt, Germany) and the lines LN405 and U343 were obtained species, and protection against advanced glycation end prod- from the “Deutsche Sammlung von Mikroorganismen und ucts and lipid peroxidation (for a comprehensive review see Zellkulturen” (DMSZ; Braunschweig, Germany). Primary Boldyrev et al. 2013). Furthermore, we and others reported cell cultures were established from tissue samples obtained the antineoplastic eff of carnosine on cancer cells from during standard surgery performed at the Neurosurgery different origin such as glioblastoma (Renner et al. 2008), Department of the University Hospital Leipzig during 2011 colon (Horii et al. 2012; Iovine et al. 2014), gastric (Shen and 2015 (see Table 1 for age and gender of patients). All et al. 2014), and cervix carcinoma (Ditte et al. 2014). patients provided written informed consent according to the In Europe, glioblastoma is the most common and in the German laws as confi med by the local committee. All glio- United States the second most common primary tumor of blastoma samples were diagnosed and have been approved the central nervous system (Ostrom et al. 2016; Sant et al. by the Neuropathology Department of the Leipzig Univer- 2012). Under the currently recommended therapy consisting sity Hospital. Primary cultures from glioblastoma tissue of surgical removal of the tumor, radiotherapy, and adju- were established as described before (Renner et al. 2008). vant chemotherapy with temozolomide, median survival of Briefly, tissue specimens from the tumor were cut into patients is only 14.6 months (Stupp et al. 2005). Therefore, approximately 1 mm3 large pieces and then separately placed new therapeutic options are urgently needed. Although car- into 25 mm2 culture fl s (TPP, Trasadingen, Switzerland) nosine has been intensively discussed as a potential anti- until tumor cells grew out. When more than 90% confl tumor drug (Gaunitz et al. 2015; Gaunitz and Hipkiss 2012; was reached, specimens were removed and primary cell cul- Hipkiss and Gaunitz 2014), up to now, the molecular mech- tures were transferred into 75 mm2 culture fl s (TPP) for anisms of carnosine’s antineoplastic eff are not com- further cultivation. Cell cultures were maintained in high pletely understood. In addition, it is not known whether the glucose DMEM (Dulbecco’s Modifi Eagle Medium with

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Carnosine’s inhibitory effect on glioblastoma cell growth is independent of its cleavage 763

Table 1 Patients of primary glioblastoma cell cultures the determination of viability after 48 h of incubation. For Patient Age (years) Gender Passage Experiments testing the eff of the CN2 inhibitor bestatin (also known as Ubenimex) on viability of cells from the glioblastoma CBA/ HPLC-MS lines G55T2 and LN405, the cells received 0 mM or 50 mM qRT- PCR carnosine and different concentrations of bestatin (0 µM, 10 µM, 50 µM, or 100 µM). In these cells viability was deter- P0052 69 Male 6 x mined after 24, 48, and 72 h of incubation. Cells used for the P0076 51 Female 4 x determination of viability after 48 and 72 h received fresh P0082 52 Male 5 x medium with the corresponding compounds after 24 h and P0086 59 Male 5 x 48 h to account for a possible instability of bestatin in the P0087 76 Male 7 x medium. After incubation, the CellTiter-Glo Luminescent P0091 79 Male 21 x Cell Viability Assay (CTG, Promega, Mannheim, Germany) P0109 55 Female 6 x was employed to determine viable cells by measuring ATP P0138 80 Female 5–10 x x in cell lysates and the CellTiter-Blue Cell Viability Assay P0167 60 Female 4 x (CTB, Promega) was used to quantify the metabolic capac-

P0174 61 Female 4 x ity in living cells. All assays were carried out according to P0223 64 Male 3 x manufacturer’s protocols. Luminescence and fl escence P0233 53 Male 3–4 x x were measured using a SpectraMax M5 multilabel reader

P0240 55 Male 2 x (Molecular Devices, Biberach, Germany). P0244 63 Female 3–6 x x P0250 79 Female 3 x Real‑time quantitative polymerase chain reaction P0258 56 Female 3 x P0297 77 Female 2–3 x x Real-time quantitative polymerase chain reaction (qRT- P0306 75 Male 3–9 x x PCR) experiments were carried out as described (Letzien P0310 71 Female 5 x et al. 2014). Briefl , 106 cells were seeded in 10 mL of P0336 54 Male 5 x medium into 10-cm cell culture dishes (TPP, Trasadingen, P0355 58 Male 4 x Switzerland) with 10 mL culture medium. After 24 h of Primary cultures of glioblastoma cells established from freshly iso- incubation, cells received fresh medium without test com- lated tumor tissue. Table shows the patient ID with the corresponding pounds or containing 25 mM L-histidine or 50 mM carno- age at time of the operation, gender and cell culture passages used sine. RNA was isolated after 24 h of incubation using the for the experiments of this study. Primary cell cultures used for cor- miRNeasy mini kit (Qiagen, Hilden, Germany) according to responding experiment are marked with “x” manufacturer’s instructions. The RNA was stored at − 80 °C qRT-PCR quantitative reverse transcription polymerase chain reaction, CBA cell based assays, HPLC-MS high-performance liquid chro- until further use. 500 ng of RNA was used for reverse tran- matography coupled with mass spectrometry scription employing the ImProm-II™ Reverse Transcrip- tion System (Promega, Mannheim, Germany) according to manufacturer’s instructions using random primer sets. DNA 4.5 g glucose/mL) supplemented with 2 mM GlutamaxTM, amplifi was performed on a Rotor-Gene 3000 system 1% penicillin/streptomycin (all from Gibco Life Technolo- (Qiagen) employing SYBR Green (Maxima SYBR Green/ gies, now Thermo Fisher Scientific, Darmstadt, Germany) ROX qPCR Master Mix, Thermo Scientific). Copy num- and 10% Fetal Bovine Serum (FBS; Biochrom GmbH, Ber- bers of individual mRNAs were determined using linearized lin, Germany), further referred to as “culture medium”, and plasmid DNA (described in Letzien et al. 2014) containing kept in incubators (37 °C, 5% CO2/95% air). the corresponding target sequence. The relative expression of pyruvate dehydrogenase kinase 4 (PDK4), serum car- Cell based assays nosinase (CNDP1) and cytosolic non-specific dipeptidase (CNDP2) was obtained by normalization to the copy num- For cell viability assays, cells were counted and seeded ber of the mRNA encoding the TATA box-binding protein into sterile 96-well plates (µClear, Greiner Bio-One, Fric- (TBP) which was used as reference gene. Data analysis was kenhausen, Germany) at a density of 5000 cells/well in performed using the RotorGene 6 software and all amplifi - 200 µL culture medium (for passage number of primary tion reactions were controlled for the appropriate products cultured cells refer to Table 1). After 24 h of cultivation by melting curve analysis. The following primer sequences (37 °C, 5% CO2/95% air), the medium was removed and were used: For CNDP1: CNDP1 forward primer: 5ʹ GAA replaced with fresh medium (100 µL/well) containing carno- GAA TAC CGG AAT AGC AG 3ʹ and CNDP1 reverse sine (10, 25, 50, 75 mM) or L-histidine (10, 25, 50 mM) for primer: 5ʹ CGG CCA GGT ATG ACT GTT 3ʹ; for CNDP2:

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CNDP2 forward primer: 5ʹ AGA AGC CCT GCA TCA CCT adding primary antibodies diluted in TBST (mouse anti AC 3′ and CNDP2 reverse primer: 5ʹ CCA CCA AAG AGC CNDP2 clone AT15E5 [Abnova; MAB11202] 1:1000; rab- CCA TC 3ʹ; for PDK4: PDK4 forward primer: 5′ CTG TGA bit anti ACTB [abcam; ab8227] 1:5000) followed by incu- TGG ATA ATT CCC 3′ and PDK4 reverse primer: 5′ GCC bation of 1 h at room temperature under constant shaking. TTT AAG TAG ATG ATA GCA 3′. For the reference gene The primary antibodies were removed by three washes with TBP: TBP forward primer: 5′ TTG ACC TAA AGA CCA TBST and a mixture of two secondary antibodies (anti-rab- TTG CAC 3′ and TBP reverse primer: 5′ GCT CTG ACT bit-IRDye680 [LI-COR; 925-68071] 1:8000; anti-mouse- TTA GCA CCT GTT 3′; (all from Biomers, Ulm, Germany). IRDye800 [LI-COR; 925-32210] 1:8000) diluted in TBST was added to the membrane. After 1 h incubation at room Western blot temperature and constant shaking, secondary antibodies were removed by three washes with TBST. The membrane For isolation of protein, 106 glioblastoma cells were seeded was dried overnight between two Whatman fi papers. into 10-cm cell culture dishes (TPP, Trasadingen, Switzer- Membranes were scanned using an Odyssey Imaging Sys- land) with 10 mL medium. After 24 h cells received fresh tem (LI-COR, Bad Homburg, Germany) and band intensities media and were subjected to additional 24 h of incubation. were determined by the Image Studio 5 software (LI-COR). Then, cells were washed twice with ice-cold washing buff

(137 mM NaCl, 5.4 mM HCl, 0.41 mM MgSO4, 0.49 mM Determination of intracellular L‑histidine MgCl2, 0.126 mM CaCl2, 0.33 mM Na2HPO4, 0.44 mM KH2PO4, 2 mM HEPES, pH 7.4) and fi y collected in To quantify intracellular amounts of histidine, a modifi 1 mL washing buff . After a brief centrifugation (5 min, method of Csámpai et al. (2004) was used. Cells from ten 500×g, 4 °C), and cells were resuspended in 150 µl of ice- glioblastoma lines and from fi e primary cultures were cold radioimmunoprecipitation assay buff (RIPA buff : seeded at a density of 300,000 cells per well into a 6-well- 50 mM Tris, 150 mM NaCl, 0.25% sodium deoxy cholate, plate in 2 mL of culture medium. After 24 h of cultivation, 0.1% SDS, 1% Nonidet P40) supplemented with PhosS- the culture medium was removed and replaced with fresh tOP™ and an in-house protease inhibitor cocktail (0.025 g/L medium containing 50 mM carnosine or 25 mM L-histidine aprotinin, 0.025 g/L leupeptin, 0.01 g/L pepstatin A, 1 mM or no compound (control). Then, cells were incubated for dithiothreitol, 2.5 mM phenylmethylsulfonylfl ide, and additional 24 h before they were washed thrice with 1 mL 2.5 mM benzamidine). After 10 min of incubation on ice, ice-cold washing buff followed by extraction using 400 µL cells were lysed by sonifi (Bioruptor, Diagenode, Sera- ice-cold methanol. After 10 min of gentle shaking on ice, ing, Belgium; settings: power: high, interval: 0.5, time: 7 extracts were collected in 1.5 mL Eppendorf-vials, wells min). The resulting cell fraction was centrifuged (5 min at were rinsed twice with 400 µL high-quality water (Milli-Q) 4 °C and 5500×g) and the resulting supernatant was trans- and 800 µL obtained were combined with the fi t 400 µL. ferred into a new 1.5 mL reaction vial and stored at − 80 °C Samples were evaporated to dryness by lyophilization (Mar- until further use. Protein concentration was determined using tin Christ Gefriertrocknungsanlagen, Osterode, Germany). the Pierce 660 nm-assay reagent according to manufacturer’s For derivatization, the freeze-dried extracts were dissolved instructions. Electrophoresis was performed using a vertical in 100 µL high-quality water (Milli-Q) and 0.5% (w/v) electrophoresis unit (Mini-Protean-Cell, Bio-Rad, Munich, ortho-phthalaldehyde (dissolved in 100 µL methanol) was Germany) and separation was carried out in 12% polyacryla- added. Derivatization was carried out at 37 °C for 45 min, mide gels. Before loading onto the gel, 30 µg protein was followed by the addition of 800 µL 0.1% formic acid (in mixed with 4 µL sample buffer (0.5 mM Tris, 40% glycerol, HPLC grade water). The obtained solution (200 µL) was 275 mM sodium dodecyl sulfate, 0.125% bromophenol blue, transferred into 250 µL conic glass inserts of 2 mL ND10 20% 2-mercaptoethanol, pH 6.8) and volume was adjusted vials, followed by high-performance liquid chromatography to 16 µL with double distilled water, followed by denatura- coupled to mass spectrometry (HPLC-MS) with 100 µL of tion (5 min, 95 °C). Separated proteins were transferred sample. overnight at 4 °C onto a low fl escence polyvinylidene fl ide membrane (ab133411 Abcam, Cambridge, United HPLC‑MS set up and data analysis Kingdom) using a wet blot system (Mini Trans-Blot Cell, Bio-Rad) with BSN-buff (48 mM Tris, 39 mM glycine, The system used for detection was an Agilent HPLC 1100 20% methanol). After transfer, membranes were blocked for (Agilent, Waldbronn, Germany) consisting of a variable 1 h at room temperature under constant shaking in Tris- wavelength detector (VWD), a well plate auto sampler and a buffered saline with Tween20 (TBST: 20 mM Tris, 134 mM binary pump, coupled with a Bruker Esquire 3000 plus elec- NaCl, 0.1% Tween 20, pH 7.6) supplemented with 2% (w/v) trospray ionization mass spectrometer run by Esquire Con- bovine serum albumin. Then, the solution was exchanged, trol 5.3 (Bruker, Bremen, Germany). The employed column

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Carnosine’s inhibitory effect on glioblastoma cell growth is independent of its cleavage 765

was a Phenomenex Gemini 5 µ C18 110 Å, 150 mm× 2 mm signifi Correlation analysis (Pearson correlation coef- with a 2 mm guard column of the same material (Phenom- fi and Kruskal–Wallis–ANOVA were carried out by enex Ltd., Aschaff g, Germany). The eluent system OriginPro 2017G (OriginLab Corporation, Northampton, consisted of two solvents, with eluent A: 0.1% formic acid in USA; Version: 2017G 64-bit SR1). acetonitrile and eluent B: 0.1% formic acid in HPLC grade water. Mobile phase fl w rate was 0.5 mL/min with the following gradient for separation: 0–10 min 90% B, 90–0% Results B within 15 min, 25–35 min 0% B, 0–90% B within 5 min and 40–47 min 90% B for column equilibration. The mass Cell viability in cultured primary glioblastoma cells spectrometer operated in positive mode (target mass: m/z and cell lines derived from glioblastoma cultivated 300; scan range: m/z 70–400), the dry gas temperature was in medium with different concentrations of carnosine set to 360°C (with a fl w rate of 11 L/min; nebulizer: 70 and L‑histidine psi). Data were analyzed using OpenChrom version 2.0.103. v20150204-1700 (Wenig and Odermatt 2010). Histidine was First, we analyzed the viability of ten cell lines and 21 pri- identifi by an authentic standard. The selective ion chro- mary cell cultures derived from glioblastoma after 48 h matogram of m/z = 272 was used for quantifi by peak of incubation in the presence of various concentrations of integration. carnosine (0, 10, 25, 50, and 75 mM) and L-histidine (0, 10, 25, and 50 mM). As can be seen in Fig. 1, viability Data presentation and statistical analysis of glioblastoma cells was significantly reduced by a concentration of 50 (p = 1.45 × 10−9) and 75 (p = 5.19 × 10−22) If not stated otherwise, data are presented as mean ± stand- mM carnosine as determined by the CTB and at a concen- ard deviation (SD). For pairwise comparisons, Welch’s t test tration of 25 (p = 2.8 × 10−2), 50 (p = 7.44 × 10−8) and 75 (unpaired two-sample test with unequal variances) was per- (p = 3.63 × 10−14) mM carnosine as determined by the CTG formed using the algorithm implemented in Excel (Micro- assay (for the individual responses of all cultures see sup- soft, Redmond, USA; Version: 14.0.7212.5000 32-Bit). For plemental Fig. 1–e). With regard to L-histidine, even the multivariate statistical analysis, a Kruskal–Wallis–ANOVA lowest concentration employed (10 mM) was able to signifi- was performed, followed by a Welch’s t test for pairwise cantly reduce the relative amount of ATP (p = 2.13 × 10−2) comparisons. To consider the false discovery rate of multiple and dehydrogenase activity (p = 6.77 × 10−4) in the tested comparisons, p values were adjusted according to Benjamini glioblastoma cells. In the presence of 25 mM (CTB: and Hochberg (1995), and a value < 0.05 was presumed to be p = 3.94 × 10−2; CTG: p = 5.53 × 10−3) and 50 mM (CTB:

Fig. 1 Viability of primary glioblastoma cell cultures and glioblas- ments of an individual culture which was normalized to the untreated toma cell lines under the influence of carnosine and L-histidine. Pri- control (set to 100%). Dark gray dots represent cell lines treated with mary glioblastoma cell cultures (21) and glioblastoma cell lines (10) carnosine or histidine, and light gray dots represent primary cell cul- were exposed for 48 h to different concentrations of carnosine (0, 10, tures treated with carnosine or histidine. Statistical significance com- 25, 50 or 75 mM) or L-histidine (0, 10, 25, or 50 mM). Viability was pared to untreated control and between equal concentrations of carno- determined by assessing metabolic activity (CTB; a) and by measur- sine and L-histidine was determined by Welch’s t test with: *p < 0.05; ing the amount of ATP in cell lysates (CTG; b). Each dot within the **p < 0.005; ***p < 0.0005. The individual results of each cell culture boxplot represents the mean obtained from six independent measure- are presented in Supplemental Fig. 1a–e

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−3 −3 p = 1.49 × 10 ; CTG: p = 2.54 × 10 ) of L-histidine, cells between protein and mRNA expression (Pearson correlation; from lines revealed a signifi y stronger loss of viability r = − 5.68 × 10−4; p = 0.999). than primary cultured cells. Overall, comparing equal concentrations, L-histidine was 1.37 ± 0.14-fold (median) more Cell viability under the influence of carnosine eff e than carnosine in decreasing cell viability. and bestatin

Next, we asked, whether the CN2 inhibitor bestatin attenu- Expression of serum carnosinase and cytosolic ates the eff of carnosine on cell viability. In Fig. 4, viabil- non‑specific dipeptidase in human glioblastoma ity in the presence of 50 mM carnosine and diff ent con- cells centrations of bestatin are presented, relative to viability of cells with only the indicated concentrations of bestatin but As the experiments presented in the previous section suggest without carnosine (set as 100% for each concentration of that carnosine may need fi t to be cleaved to L-histidine and bestatin). As illustrated, bestatin does not attenuate the eff β-alanine to deploy its eff we asked whether the glioblas- of carnosine in both cell lines, independent of the employed toma cells express the mRNA of the required enzymes at all. concentrations of bestatin and the time of measurement. Therefore, we analyzed CNDP1 and CNDP2 expression in Only at concentrations of 50 µM and 100 µM bestatin and 31 glioblastoma cell cultures by qRT-PCR. As can be seen in the case of LN405 with regard to ATP in cell lysates after in Fig. 2, CNDP2 was expressed in all analyzed samples 72 h of incubation, the eff of carnosine on viability was with a relative expression ranging from 0.59 to 30.24 copy statistically not signifi nt (p = 0.31 and 0.33, respectively). numbers per TBP copy (note that TBP mRNA has ~ 100 However, this was most likely caused by an already strong copies per ng RNA). In contrast to that, CNDP1 was only eff of bestatin on viability after 72 h exposure even in weakly expressed or not detectable with a relative expres- the absence of carnosine which resulted in a high standard sion ranging from 0 to 0.14 copy numbers per TBP copy. deviation (50 µM: 42 ± 17% viability without and 27 ± 13% Next, we asked whether the presence of CNDP2 mRNA in with carnosine, 100 µM: 26 ± 16% viability without, and glioblastoma cells also results in a corresponding protein 13 ± 7% with carnosine). At this point, it has to be noted that expression. In the western blot presented in Fig. 3, it can we observed a negative eff on viability with increased be seen that CN2 protein was detectable in all investigated concentrations of bestatin and exposure time, even in the samples, suggesting the possibility that carnosine could absence of carnosine (see supplemental Fig. 2). More impor- indeed be cleaved intracellularly. Noteworthy, by quanti- tant, comparing viability in the presence of carnosine and fying CN2 band intensity, we could not fi a correlation diff ent concentrations of bestatin and incubation time does

Fig. 2 Expression of CNDP1 and CNDP2 mRNA in primary glioblastoma cell cultures and glioblastoma cell lines. Total RNA from 21 primary glioblastoma cell cultures and ten glioblastoma cell lines was extracted, reverse transcribed and analyzed by qRT-PCR. CNDP1 and CNDP2 mRNA copy numbers were determined using standards and relative expression was calculated by the normalization to the TBP mRNA copy number of each sample. Data are represented as mean and standard deviation of three measurements. An x-axis break has been used because of the high differences between the absolute expression of CNDP1 and CNDP2 mRNA

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Carnosine’s inhibitory effect on glioblastoma cell growth is independent of its cleavage 767

Fig. 3 Expression of CN2 protein in glioblastoma cell lines. Protein from ten glioblastoma cell lines was isolated and subjected to SDS-PAGE. Western blotting was performed using antibodies against CN2 (53 kDa) and β-actin (42 kDa). Upper panel: western blot image as analyzed by an Odyssey Imaging System. Lower panel: quantitative analysis of bands from the upper panel determined by Image Studio 5

not indicate an infl of the CN2 inhibitor on carnosine’s L-histidine (a concentration resulting in a similar decrease eff on viability. of viability as observed in the presence of 50 mM carnosine, Fig. 1), we detected a ~ 40-fold higher abundance Comparison of intracellular L‑histidine in primary of intracellular L-histidine compared to incubation in the cultured glioblastoma cells and cell lines derived presence of 50 mM carnosine (Fig. 5). This observation from glioblastoma exposed to extracellular carnosine also confi ms the notion that cleavage of carnosine is not required for its antineoplastic eff The experiments presented in the previous section indicated that a release of L-histidine may not be required for the antineoplastic eff of carnosine. However, as via- Expression of PDK4 mRNA under the influence bility was obviously negatively aff by the inhibitor of carnosine and L‑histidine and correlation bestatin itself, these experiments do not unequivocally between viability and influence on expression demonstrate that the antineoplastic eff of carnosine is independent of the release of L-histidine in the absence Finally, we investigated whether the eff of L-histidine of the inhibitor. Therefore, we investigated whether the (25 mM) and carnosine (50 mM) on viability correlates release of L-histidine and the effect of carnosine on viabil- with their infl on PDK4 expression. As can be seen ity show any correlation. As can be seen in Fig. 5a, except in Fig. 6a, carnosine signifi y induced the expression for P0297 and G55T2, treatment with 50 mM carnosine of PDK4 mRNA in eight samples, whereas a signifi y resulted in a signifi 1.22 to 2.5-fold increase of the reduced expression was found in four samples. Under the intracellular abundance of histidine (with p values reach- infl of L-histidine, PDK4 mRNA expression was sig- ing from 3.12 × 10−4 to 7.12 × 10−3). This indicates that the nifi y induced in 21 samples. Comparing the eff of dipeptide is cleaved to a certain amount inside the cells. the two compounds on PDK4 expression (Fig. 6b), 18 cell However, there was no correlation between the expres- cultures responded in the same way (both carnosine and sion of CN2 (Fig. 3) and the relative release of L-histidine. L-histidine induced or reduced PDK4 mRNA expression This is seen, for example, in the low expression of CN2 in when treatment of at least one compound was signifi y MZ18 compared to its higher expression in LN229, which diff ent compared to the untreated control), whereas six is not refl ed by a corresponding diff rence in L-histidine cell cultures responded in an opposite way. Furthermore, release. More important, we did not see any signifi t there was a signifi correlation between the infl correlation between the decrease of viability under the of carnosine and L-histidine on PDK4 mRNA expression infl of 50 mM carnosine and the relative release of (Pearson correlation; r = 0.419; p = 0.019). Comparing the L-histidine (supplemental Fig. 3). This also indicates that eff on PDK4 expression with the eff on viability, no the antineoplastic eff is independent of the cleavage correlation was found neither for L-histidine (Fig. 6c) nor of the dipeptide. Analyzing the intracellular abundance for carnosine (Fig. 6d). of L-histidine in the presence of 25 mM extracellular

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Fig. 4 Viability of G55T2 and LN405 glioblastoma cells under the 72 h of incubation, viability was determined by determining meta- influence of carnosine and the dipeptidase-inhibitor bestatin. Via- bolic activity [dehydrogenase (DH) activity] and by measuring the bility of cells from the lines G55T2 and LN405 in the presence of amount of ATP in cell lysates (ATP in cell lysates). Data are repre- 50 mM carnosine and different concentrations of bestatin (0, 10, 50, sented as mean and standard deviation of six independent measure- or 100 µM) was compared to the viability of cells with the inhibitor ments. Statistical significance was determined by Welch’s t test with: bestatin but without carnosine, setting the viability without carnosine *p < 0.05; **p < 0.005; ***p < 0.0005 for each concentration of bestatin to 100 percent. After 24, 48, and

Discussion cell cultures and ten cell lines originated from glioblastoma, we observed that with regard to the eff or concentrations The antineoplastic effect of carnosine, which was first required to induce similar losses of viability, L-histidine described by Nagai and Suda (1986), has been confi med was more eff e at lower concentrations than carnosine. in vitro (Ditte et al. 2014; Iovine et al. 2014; Renner et al. We also observed increased concentrations of intracellular 2008; Shen et al. 2014) and in vivo (Horii et al. 2012; L-histidine when cells were exposed to carnosine. As we Renner et al. 2010) by several research groups. Hence, identifi the presence of CN2, we assume that this intra- its potential as an anti-tumor agent has been discussed in cellular L-histidine is produced by cleavage of carnosine a number of review articles (Gaunitz et al. 2015; Gaunitz catalyzed by the enzyme. Comparing CN2 expression with and Hipkiss 2012; Hipkiss and Gaunitz 2014). As we pre- the amount of intracellular L-histidine, we found no correla- viously demonstrated that L-histidine is able to mimic the tion. However, at this point, it has to be taken into account antineoplastic eff of the dipeptide (Letzien et al. 2014), that because of diff ent cell sizes, it is almost impossible we wondered whether carnosine has to be cleaved to inhibit to precisely compare the exact changes of intracellular con- tumor cell growth. Comparing the viability of 21 primary centrations of L-histidine between diff ent cells. However,

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Carnosine’s inhibitory effect on glioblastoma cell growth is independent of its cleavage 769

Fig. 5 Intracellular abundance of histidine in glioblastoma cells incu- gation) compared to untreated control cells from six independent bated in the presence of 50 mM carnosine and 25 mM histidine. a experiments. Statistical significance was determined by Welch’s t test Cells from ten lines (dark gray) and five primary glioblastoma with: *p < 0.05; **p < 0.005; ***p < 0.0005. b Relative intracellular cell cultures (light gray) were exposed for 24 h to 50 mM carnos- abundance of L-histidine in cells from 10 cell lines incubated in the ine. Then, amino acids were extracted and analyzed by HPLC-MS. presence of 50 mM carnosine or 25 mM L-histidine. Each dot rep- Changes of the relative intracellular abundances of L-histidine are resents the mean obtained from six independent measurements of an shown as mean percent and standard deviation (using error propa- individual culture cleavage appears to be weak as the intracellular concentra- (74 ± 18%), whereas a concentration of 198 mM carnosine tion of L-histidine in the presence of 25 mM L-histidine in resulted in only 39 ± 3% LDH release compared to 20 ± 5% the medium is ~ 40-times higher than the amount of L-histi- LDH release in untreated control cells. This also points to dine in cells incubated in the presence of 50 mM carnosine. the possibility that other mechanisms are responsible for the At this point, it also has to be noted that Teufel et al. (2003) increased toxicity of L-histidine than those responsible for claimed that the intracellular pH is far from the optimal pH the specifi antineoplastic eff of carnosine. At this point, required for effective cleavage of carnosine by CNDP2. A it should also be noted that we recently demonstrated that very low release of L-histidine from carnosine has also been in co-cultures of tumor cells with non-tumor cells, carnos- demonstrated by Son et al. (2008), investigating the intracel- ine selectively eliminated the tumor cells (Oppermann et al. lular concentration of amino acids of Caco-2 cells cultivated 2018). In these experiments, in which the cells were incu- in the presence of 50 mM carnosine for 6 and 27 h. In these bated up to several weeks, lower concentrations of carnosine experiments, the intracellular amount of L-histidine in the have been employed than those that were used in the experi- presence of carnosine was more than 50 times lower than ments presented in the present study. This has to be noted, as that observed in the presence of 50 mM L-ala-L-his. we are aware that it may be diffi to achieve the compound Obviously, carnosine does not need to be cleaved into its concentrations employed in the present investigation in vivo. single amino acids to deploy its antineoplastic eff but the On the other hand, it is also diffi to estimate, which intra- question remains, whether L-histidine bound to the β-alanine cellular concentrations may be achievable when carnosine moiety in carnosine is suffi to induce the antineoplastic is delivered orally, as we do not know whether carnosine eff of carnosine and whether β-alanine may be substi- can effi y be protected from degradation by CN1 (see tuted by another moiety. This question has to be elaborated below). We also do not know whether it can be transported by further experiments, as it bears the interesting aspect to the target cells, whether it may be resynthesized from that it could lead to the development of anti-cancer drugs the single amino acids, and whether it may accumulate in which may have an advantage over carnosine. It also has to cells which could take it up. Anyway, the most frequently be asked whether L-histidine could be used as a supplement discussed obstacle using carnosine as an orally given drug is instead of carnosine. As demonstrated by cell injury assays its rapid degradation by serum carnosinase in humans (Len- with cultivated rat hepatocytes, this appears to be no good ney et al. 1982). Despite the observation that the dipeptide choice (Rauen et al. 2007). In these experiments, highly is eff ctive as a drug in different diseases (Baraniuk et al. increased lactate dehydrogenase (LDH) release, as an indica- 2013; Boldyrev et al. 2008; Chengappa et al. 2012; Chez tor of cellular necrosis, was seen in the presence of 198 mM et al. 2002) and despite the fact that it may be protected (92 ± 1%), 76 mM (91 ± 1%), and 50 mM L-histidine by deposition in liver (Gardner et al. 1991) or erythrocytes

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770 H. Oppermann et al.

Fig. 6 Expression of PDK4 mRNA after exposure to carnosine or untreated control (set to 1). Statistical significance was determined by L-histidine. 21 primary glioblastoma cell cultures and ten glioblas- Welch’s t test with: *p < 0.05; **p < 0.005; ***p < 0.0005. b Com- toma cell lines were incubated in the presence of 50 mM carnosine, parison of PDK4 mRNA expression under the influence of carnosine 25 mM L-histidine, or vehicle control for 24 h. Then, total RNA was and L-histidine. The data presented in a are here depicted with fold extracted, reverse transcribed and analyzed by qRT-PCR. PDK4 increase of relative PDK4 mRNA expression under the influence car- mRNA copy numbers were determined using standards and relative nosine on the x-axis, and with fold increase under the influence of expression was calculated by the normalization to the TBP mRNA L-histidine on the y-axis. c Comparison between viability and PDK4 copy number of each sample. a Fold enhancement of PDK4 expres- mRNA expression under the influence of 25 mM L-histidine. d Com- sion compared to untreated control cells. Data are represented as parison between viability and PDK4 mRNA expression under the mean and standard deviation of three measurements compared to the influence of 50 mM carnosine

(Chaleckis et al. 2016), the high activity of CN1 in human blood–brain barrier in case of brain tumors, and are selec- serum will certainly strongly aff the eff e concentra- tive in preventing cancer cell proliferation. For the purpose tion achievable in a patient’s tumor tissue when given orally. of further drug development, it is also of major importance In addition, it has to be asked whether cleavage of carnosine to understand the molecular mechanisms responsible for the by CN1 may increase serum concentrations of L-histidine to antineoplastic eff hepatotoxic concentrations. Therefore, it would be interest- Aside from a plethora of reports on carnosine’s infl - ing to analyze whether other L-histidine- or imidazole-con- ence on signaling molecules in diff ent models (for review, taining compounds which are not degradable by CN1 and see Gaunitz et al. 2015), the currently best described eff are not toxic to other cells, may be useful alternatives. How- of carnosine on transcription in glioblastoma cells is its ever, it has to be taken into account that these compounds infl on transcription of PDK4 (Letzien et al. 2014). need to be able to be taken up by tumor cells, can pass the Therefore, we also investigated whether we could see any

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Carnosine’s inhibitory effect on glioblastoma cell growth is independent of its cleavage 771

correlation between carnosine and L-histidine regarding Bellia F, Vecchio G, Rizzarelli E (2014) Carnosinases, their substrates their eff on PDK4 mRNA expression (Fig. 6). As we and diseases. Molecules 19:2299–2329. https://doi.org/10.3390/ molecules19022299 could demonstrate that carnosine and L-histidine infl Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: PDK4 mRNA expression in 18 out of 31 cell cultures in the a practical and powerful approach to multiple testing. J R Stat Soc same way, this may indicate that at least in some cultures Ser B (Methodological) 57:289–300 L-histidine and carnosine share similarities with regard to Boldyrev A, Fedorova T, Stepanova M, Dobrotvorskaya I, Kozlova E, Boldanova N, Bagyeva G, Ivanova-Smolenskaya I, Illarioshkin their infl on transcription. Although this only moder- S (2008) Carnosine [corrected] increases efficiency of DOPA ate correlation may be tempered by a limited sample size, therapy of Parkinson’s disease: a pilot study. Rejuvenation Res it is at least obvious that there is no correlation between 11:821–827. https://doi.org/10.1089/rej.2008.0716 carnosine’s and L-histidine’s eff on viability and expres- Boldyrev AA, Aldini G, Derave W (2013) Physiology and pathophysiology of carnosine. Physiol Rev 93:1803–1845. https://doi. sion of PDK4 mRNA. Therefore, we conclude that changes org/10.1152/physrev.00039.2012 of PDK4 expression may not be responsible for carnosine’s Chaleckis R, Murakami I, Takada J, Kondoh H, Yanagida M (2016) antineoplastic eff Individual variability in human blood metabolites identifi age- In summary, we demonstrate that the cleavage of car- related diff ences. Proc Natl Acad Sci USA 113:4252–4259. https://doi.org/10.1073/pnas.1603023113 nosine and the release of L-histidine is not required for the Chengappa KR, Turkin SR, DeSanti S, Bowie CR, Brar JS, Schlicht PJ, antineoplastic eff of the dipeptide, which can be deduced Murphy SL, Hetrick ML, Bilder R, Fleet D (2012) A preliminary, from three observations: (1) The CN2 inhibitor bestatin does randomized, double-blind, placebo-controlled trial of L-carnosine not attenuate the eff of carnosine on tumor cell viability. to improve cognition in schizophrenia. Schizophr Res 142:145– 152. https://doi.org/10.1016/j.schres.2012.10.001 (2) Intracellular cleavage of carnosine is observable, but Chez MG, Buchanan CP, Aimonovitch MC, Becker M, Schaefer K, has a low effi . (3) Roughly 40-times higher amounts Black C, Komen J (2002) Double-blind, placebo-controlled study of intracellular free L-histidine in the presence of 25 mM of L-carnosine supplementation in children with autistic spectrum extracellular L-histidine have the same eff on tumor cell disorders. J Child Neurol 17:833–837 Csámpai A, Kutlán D, Tóth F, Molnár-Perl I (2004) O-Phthaldialde- viability as concentrations of free L-histidine observed in the hyde derivatization of histidine: stoichiometry, stability and reac- presence of 50 mM extracellular carnosine. tion mechanism. J Chromatogr A 1031:67–78 Ditte Z, Ditte P, Labudova M, Simko V, Iuliano F, Zatovicova M, Csa- Acknowledgements We would like to thank Flamma [Flamma s.p.a. derova L, Pastorekova S, Pastorek J (2014) Carnosine inhibits Chignolo d’Isola, Italy (https://www.fl agroup.com)] for the gener- carbonic anhydrase IX-mediated extracellular acidosis and sup- ous supply with very high-quality carnosine for all of our experiments. presses growth of HeLa tumor xenografts. BMC Cancer 14:358. In addition, we would like to thank Dr. Hans-Heinrich Foerster from https://doi.org/10.1186/1471-2407-14-358 the Genolytic GmbH (Leipzig, Germany) for genotyping and confi ma- Drozak J, Chrobok L, Poleszak O, Jagielski AK, Derlacz R (2013) tion of cell identity and last not least Mrs. Susan Billig for technical Molecular identifi of carnosine N-methyltransferase as assistance. chicken histamine N-methyltransferase-like protein (hnmt-like). PLoS One 8:e64805. https://doi.org/10.1371/journal.pone.00648 Author contributions KP performed most of the experiments with 05 contributions of HO and RB-S. CB established the HPLC-MS method Gardner MLG, Illingworth KM, Kelleher J, Wood D (1991) Intestinal- with contributions of HO and performed the HPLC-MS measurements. absorption of the intact peptide carnosine in man, and comparison JM did the surgery and revised the manuscript. HO and FG designed with intestinal permeability to lactulose. J Physiol 439:411–422 the study and wrote the manuscript. All authors read and approved the Gaunitz F, Oppermann H, Hipkiss AR (2015) Carnosine and cancer. manuscript. In: Preedy VR (ed) Imidazole dipeptides. The Royal Society of Chemistry, Cambridge, pp 372–392 Gaunitz F, Hipkiss AR (2012) Carnosine and cancer: a perspec- Compliance with ethical standards tive. Amino Acids 43:135–142. https://doi.org/10.1007/s0072 6-012-1271-5 Conflict of interest The authors declare that they have no potential Gulewitsch W, Amiradzibi S (1900) Ueber das Carnosin, eine neue conflict of interest. organische Base des Fleischextraktes. Ber Dtsch Chem Ges 33:1902–1903 Informed consent All patients provided written informed consent Hipkiss AR, Gaunitz F (2014) Inhibition of tumour cell growth by according to German law as confi med by the local committee (#144- carnosine: some possible mechanisms. Amino Acids 46:327–337 2008) in accordance with the 1964 Helsinki declaration and its later Horii Y, Shen J, Fujisaki Y, Yoshida K, Nagai K (2012) Eff of amendments. L-carnosine on splenic sympathetic nerve activity and tumor proliferation. 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Teufel M, Saudek V, Ledig JP, Bernhardt A, Boularand S, Carreau org/10.1016/j.cca.2010.10.016 A, Cairns NJ, Carter C, Cowley DJ, Duverger D, Ganzhorn AJ, Rauen U, Klempt S, de Groot H (2007) Histidine-induced injury to Guenet C, Heintzelmann B, Laucher V, Sauvage C, Smirnova T cultured liver cells, eff of histidine derivatives and of iron (2003) Sequence identifi and characterization of human chelators. Cell Mol Life Sci 64:192–205. https://doi.org/10.1007/ carnosinase and a closely related non-specifi dipeptidase. J Biol s00018-006-6456-1 Chem 278:6521–6531 Renner C, Seyff rth A, de Arriba S, Meixensberger J, Gebhardt R, Wenig P, Odermatt J (2010) OpenChrom: a cross-platform open Gaunitz F (2008) Carnosine inhibits growth of cells isolated from source software for the mass spectrometric analysis of chro- human glioblastoma multiforme. Int J Pept Res Ther 14:127–135. matographic data. BMC Bioinform 11:405. https://doi. https://doi.org/10.1007/s10989-007-9121-0 org/10.1186/1471-2105-11-405 Renner C, Zemitzsch N, Fuchs B, Geiger KD, Hermes M, Hengstler J, Gebhardt R, Meixensberger J, Gaunitz F (2010) Carnosine retards Publisher’s Note Springer Nature remains neutral with regard to tumor growth in vivo in an NIH3T3-HER2/neu mouse model. Mol jurisdictional claims in published maps and institutional affiliations. Cancer 9:2. https://doi.org/10.1186/1476-4598-9-2 Romero SA, Hocker AD, Mangum JE, Luttrell MJ, Turnbull DW, Struck AJ, Ely MR, Sieck DC, Dreyer HC, Halliwill JR (2016)

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3.3. Supplemental materials

(also online available)

Patients and patient derived primary cultured glioblastoma cells

Label Culture Passage Sex Age MGMT IDH1/2 p53 GFAP Type P0109 08_12 6. P. f 55 GBM negativ unknown unknown positiv primary P0223 22_14 3. P. m 65 GBM negativ wild type unknown positiv primary P0076 27_11 4. P. f 51 GBM unknown unknown positiv primary P0174 39_13 4. P. f 61 GBM negativ wild type ˂5% positiv primary P0082 41_11 5. P. m 52 GBM negativ unknown ˂5% positiv primary P0086 43_11 5. P. m 59 GBM negativ unknown unknown positiv primary P0052 45_10 6. P. m 69 GBM positiv unknown unknown primary P0240 47_14 2. P. m 56 GBM positiv wild type unknown positiv primary P0244 51_14 3. P. f 64 GBM negativ wild type ˂5% positiv primary P0091 52_11 21. P. m 79 GBM unknown unknown unknown primary P0250 56_14 3. P. f 80 GBM negativ wild type 25% positiv primary P0258 65_14 3. P. f 57 GBM negativ wild type 15-20% positiv primary P0138 71_12 5. P. f 81 GBM negativ unknown unknown positiv primary P0297 106_14 2. P. f 78 GBM positiv wild type 25% positiv primary P0310 07_15 5. P. f 72 GBM positiv wild type 5-10% positiv primary P0336 31_15 5. P. m 54 GBM negativ wild type 5-10% positiv primary P0306 04_15 3. P. m 76 GBM positiv wild type 80% positiv primary P0355 105_14 4. P. m 59 GBM negativ wild type ˃50% positiv primary P0233 38_14 3. P. m 53 GBM positiv wild type 10% positiv primary P0167 29_13 9. P. f 61 GBM positiv wild type pos. positiv primary P0087 45_11 7. P. m 76 GBM unknown unknown unknown positiv primary

34 4. Summary

Dissertation zur Erlangung des akademischen Grades Dr. med.

Titel Carnosine’s inhibitory effect on glioblastoma cell growth is independent of its cleavage eingereicht von: Katharina Purcz angefertigt an: Universität Leipzig, Klinik und Poliklinik für Neurochirurgie betreut von

Prof. Dr. Frank Gaunitz

Prof. Dr. Jürgen Meixensberger

Monat und Jahr (der Einreichung): 03/2020

The naturally occurring dipeptide carnosine (ß-alanyl-L-histidine) was first isolated in 1900 [43]. 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 [64]. 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) [18]. The latter shows a widespread distribution and can be inhibited by the dipeptidase-inhibitor bestatin, whereas CN1 appears only in the human brain, kidney glomerulus, liver and serum [75, 76, 118] and is responsible for rapid degradation of carnosine in serum. The semi-essential amino acid L-histidine is supposed to be mainly responsible for the dipeptides physiological properties based on its imidazole moiety [18]. 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 [14]. Furthermore, the biogenic dipeptide regulates intracellular calcium homeostasis, acts as a pH buffer and as a metal ion chelator [8]. Based on these primary functions, the dipeptide supports mitochondrial activity and diminishes proteotoxicity [51]. Current studies mainly consider these benefits in muscle tissue [7, 53] and refer to cardiovascular [13] and neurodegenerative diseases [5, 24, 52, 80]. In 1986, Nagai and Suda first revealed tumor growth inhibition after using carnosine in a sarcoma mouse model [86]. Later, Holliday and McFarland confirmed these observations in HeLa cells in vitro [54]. Afterwards, Renner et al. demonstrated an anti- proliferative effect of carnosine on human glioblastoma cells [99, 100].

35 Unfortunately, the dipeptide’s exact molecular mechanisms on tumor cells are still not entirely understood. It has been suggested, that carnosine may influence signal transduction pathways, such as CREB-related signaling by Ca2+ activation, ERK1/2 phosphorylation or HIF-1 alpha signaling. In addition, the suppression of NF-κB/STAT1 signaling which induces cell cycle arrest has been proposed. Another unresolved question is, whether the dipeptide itself is required for the antineoplastic 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 [98]. Significant risk factors are still unknown [88]. At diagnosis, the median age is 64 years and the disease is usually found in a progressed stage [84, 98]. In a short clinical history, first symptoms are headaches, epileptic seizures, nausea, and personality changes, depending on tumor localization and size [27, 42, 66]. 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 [66]. 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 [115]. Experiments by Letzien et al. demonstrated that L-histidine mimics the anti-neoplastic effect of carnosine in three glioblastoma cell lines investigated [77]. 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. Regarding the effect of carnosine and L-histidine on PDK4 mRNA expression, similar effects were observed in 18 of 31 cell cultures. However, no correlation between viability and PDK4 expression could be found. Therefore, it may be suggested, that changes in PDK4 expression are not responsible for carnosine’s anti-proliferative impact.

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 (gene:

36 CNDP2) 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 [97]. 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

37 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 [80]. 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 [107].

Parts of the results presented in this work have previously been published in Amino Acids as a joint work with H. Oppermann, J. Meixensberger and F. Gaunitz, 03/2019 (PMID: 30863889 DOI: 10.1007/s00726-019-02713-6)

38 5. References

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46 6. Appendix

6.1. Declaration of independent work:

Erklärung über die eigenständige Abfassung der Arbeit

Hiermit erkläre ich, dass ich die vorliegende Arbeit selbstständig und ohne unzulässige Hilfe oder Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe. Ich versichere, dass Dritte von mir weder unmittelbar noch mittelbar eine Vergütung oder geldwerte Leistungen für Arbeiten erhalten haben, die im Zusammenhang mit dem Inhalt der vorgelegten Dissertation stehen, und dass die vorgelegte Arbeit weder im Inland noch im Ausland in gleicher oder ähnlicher Form einer anderen Prüfungsbehörde zum Zweck einer Promotion oder eines anderen Prüfungsverfahrens vorgelegt wurde. Alles aus anderen Quellen und von anderen Personen übernommene Material, das in der Arbeit verwendet wurde oder auf das direkt Bezug genommen wird, wurde als solches kenntlich gemacht. Insbesondere wurden alle Personen genannt, die direkt an der Entstehung der vorliegenden Arbeit beteiligt waren. Die aktuellen gesetzlichen Vorgaben in Bezug auf die Zulassung der klinischen Studien, die Bestimmungen des Tierschutzgesetzes, die Bestimmungen des Gentechnikgesetzes und die allgemeinen Datenschutzbestimmungen wurden eingehalten. Ich versichere, dass ich die Regelungen der Satzung der Universität Leipzig zur Sicherung guter wissenschaftlicher Praxis kenne und eingehalten habe.

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47 6.2. Statement of the own contribution

Mrs. Purcz performed all RT-qPCR and Western Blot experiments independently, and cell cultivation and cell-based assays partially assisted by Rainer Baran-Schmidt. In addition, she performed high-performance liquid chromatography coupled to mass spectrometry together with Henry Oppermann and under supervision by Claudia Birkemeyer. Mrs. Purcz was responsible for the analysis of all data obtained and was assisted by Henry Oppermann and Frank Gaunitz for statistical analysis. She wrote most parts of the manuscript being assisted by Henry Oppermann and Frank Gaunitz. Surgery was done by Jürgen Meixensberger and his clinical team who together with all other authors approved the published version of the manuscript.

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6.3. Acknowledgements

I would like to express my very great appreciation to Prof. Dr. Jürgen Meixensberger and Prof. Dr. Frank Gaunitz for giving me the opportunity to research and to accomplish the degree of doctorate at the Department of Neurosurgery of the University Hospital Leipzig. I am particularly grateful for the invaluable supervision by Prof. Dr. Frank Gaunitz. Without his guidance, persistent effort, and time this dissertation would not have been possible. He continually conveyed great motivation, support, and advice throughout the entire period of contriving, performing, and assessing this work. Profound thanks also for enabling me to participate in an international congress. In addition, I wish to acknowledge the help and the encouragement given by Dr. Henry Oppermann. He provided me with professional advice, as well as patient assistance in laboratory procedures, especially during the measurements and their computerized evaluation. Assistance given by Mr. Rainer Baran-Schmidt is also greatly appreciated. He introduced me to the laboratory and demonstrated the functional principals of the experiments and their analysis. I also thank Dr. Claudia Birkemeyer and Dr. Susan Billig for their attendance and their explanations during my measurements at the Institute of Analytical Chemistry of the University Leipzig. I would also like to express my deep gratitude to my family and friends for their support and their backing and to all those who have directly or indirectly guided me in writing this assignment. Furthermore, I would like to offer my special thanks to Laura Clart and Dr. Dennis Basel, for their mindful proofreading and help in language questions.