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