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Das Enzym 5-Lipoxygenase (5-LO) spielt eine essentielle Rolle in der Biosynthese der Leukotriene, bioaktiver Metabolite der Arachidonsäure (AA), die an einer Vielzahl entzündlicher und allergischer Erkrankungen beteiligt sind. Die 5-LO wird bevorzugt in Zellen myeloiden Ursprungs wie Granulozyten, Monozyten oder B-Lymphozyten exprimiert. In die Regulation der zellulären 5-LO-Aktivität in der Epstein-Barr Virus-transformierten B-lymphozytären Zelllinie BL41-E95-A sind Caspasen, Aspartat-spezifische Cysteinproteasen, involviert. Das Passagieren von BL41-E95-A führt zu einer Erhöhung der Proliferationsrate der B-Lymphozyten sowie zu einem deutlichen Verlust der 5-LO-Aktivität, der mit dem Auftreten eines 62 kDa-Spaltproduktes der 5-LO und einer signifikanten Aktivitätserhöhung der Caspase-8 und -6 korreliert. Isolierte humane 5-LO wird durch rekombinante Caspase-6 zwischen Asp170 und Ser171 zu einem 58 kDa-Fragment in vitro gespalten, wobei das Tetrapeptid VEID170 innerhalb der 5-LO als Erkennungsmotiv für den Angriff der Caspase-6 dient. In einigen weiteren untersuchten Zelllinien wie Mono Mac 6 (MM6), RBL-1, PMNL oder HeLa, die nicht den B-Lymphozyten angehören, konnte die 5-LO-Spaltung weder durch das Passagieren von Zellen noch durch die Behandlung mit diversen proapoptotischen Agentien ausgelöst werden. Laut Ergebnissen aus in vitro-Untersuchungen scheinen 5-LO-positive HeLa- bzw. MM6-Zellen einen Faktor zu exprimieren, der die 5-LO direkt oder indirekt vor dem Angriff der Caspase-6 und anschließender Prozessierung schützt. Die in den BL41-E95-A-Zellen beobachtete Aktivierung der Caspasen mit anschließender Prozessierung der 5-LO lässt sich durch zwei Pflanzeninhaltsstoffe supprimieren, das Hyperforin (HP) aus Johanniskraut-Extrakten und das Myrtucommulon (MC) aus Myrte-Blättern. Beide Verbindungen scheinen in B-Lymphozyten zu einer Hemmung der Caspasen-Aktivierung zu führen. Nichtsdestotrotz führt die Behandlung der B-Lymphozyten mit HP bzw. MC zu einem apoptotischen Tod der Zellen. Offensichtlich wird dabei ein (unbekannter) einzigartiger Mechanismus der Apoptose-Induktion ausgelöst. In der vorliegenden Arbeit konnte zum ersten Mal eine potente Apoptose-induzierende Wirkung des natürlich vorkommenden Myrtucommulons auf Krebszelllinien gezeigt werden. In allen getesteten Krebszelllinien führte Myrtucommulon zum Zelltod, wobei die HL-60-Zellen mit einem IC50-Wert von 3,26 ± 0,51 µM MC am sensitivsten gegenüber MC-Einfluss waren. Zusätzlich konnte in HL-60- und MM6-Zellen nach MC-Behandlung neben einer erhöhten Caspasen-Aktivität und PARP-Spaltung ein signifikanter DNA-Abbau detektiert werden. Von besonderer Bedeutung ist die Tatsache, dass die zytotoxische MC-Wirkung eine bemerkenswerte Selektivität für entartete Zelllinien zu besitzen scheint und gegenüber nicht-transfizierten Zellen minimal ist.
Alzheimer’s Disease (AD) is the most common neurodegenerative disorder marked by progressive loss of memory and cognitive ability. The pathology of AD is characterised by the presence of amyloid plaques, intracellular neurofibrillary tangles and pronounced cell death. The aim of this thesis was to investigate pathways involved in the Aß cascade of neurodegeneration. Since novel findings indicate that already this Aß species exerts neurotoxic effects long before hyperphosphorylated tau, neurofibrillary tangles and extracellular Aß plaques appear, the investigations were accomplished with specific regard to the effects of intracellular Aß. The Swedish double mutation in the APP gene results in six- to eightfold increased Aß production of both Aß1-40 and Aß1-42 compared to human wildtype APP cells (APPwt). Data obtained from PC12 cells indicate that it is possible to specifically increase the Aß load without enhancing APP expression levels. On the basis of these findings, it seemed possible to investigate dose-dependent effects of Aß in multiple experimental designs. These assay designs were created in order to mimick different in-vivo situations that are discussed to occur in AD patients: APPsw PC12 cells exhibit low physiological concentrations of Aß within picomolar range in contrast to APPsw HEK cells, expressing Aß levels within the nanomolar range. Of note, the APPsw HEK cells showed a specific and highly significant increase in the intracellular accumulation of insoluble Aß1-42. Moreover, an intracellular accumulation of Aß and APP was found in the mitochondria of the HEK APPsw cells suggesting a direct impact on mitochondrial function on these cells. This effect might finally lead to disturbances in the energy metabolism of the cell or to increased cell death. Furthermore, baseline g- and ß-secretase activity was assessed since these enzymes represent promising therapeutic targets to slow or halt the disease process. As expected, ß-secretase activity was significantly elevated in all APPsw cell lines. This might be due to the proximity of the Swedish double mutation next to the N-terminus of the Aß sequence. Interestingly, g-secretase activity was similarly increased in PC12 APPsw cells. In addition, the toxicity of different Aß species was investigated in SY5Y and PC12 cells with regard to their effect on cellular viability mirrored by mitochondrial activity using MTT assay. Here, it turned out that not monomers, but already dimers are neurotoxic correlates. Fibrillar Aß species showed the highest toxicity. In the next step, SY5Y cells forming endogenous, dimeric APP and Aß were investigated. In accordance with previous findings, these cells showed a decreased MTT reduction potential in comparison to APPwt and control SY5Y cells reflecting a decrease of cellular viability. The impaired energy metabolism of the cells was even more drastically mirrored by reduced baseline ATP levels. In the second part of this thesis, the expression and intracellular distribution of Bcl-2 family proteins and pro-apoptotic mitochondrial factors under baseline conditions and during oxidative stress were analyzed in the APPwt and APPsw bearing cells. The most prominent finding was the reduction of expression levels of the anti-apoptotic factor Bcl-xL in the cytosolic fractions of APPwt and APPsw PC12 cells. This might indicate that a lack of anti-apoptotic factors or their altered intracellular distribution, rather than an increase in caspase-dependent pro-apoptotic factors, could be responsible for the increased vulnerability of APPwt- and APPsw-transfected PC12 cells against oxidative stress. Since total Bcl-xL expression was unaffected in PC12 cells, in contrast to APPwt and APPsw-expressing SY5Y and HEK cells revealing significantly decreased Bcl-xL expression levels. Thus, alterations in Bcl-xL distribution seem to be an early event in the disease process. Increasing Bcl-xL expression might potentially be one promising strategy for AD modification. PC12 and HEK cells bearing APPsw or APPwt were treated with the potent g-secretase inhibitor DAPT. Of note, DAPT did not only efficiently block Aß production, but additionally led to an elevation of the MTT reduction potential, reflecting an increase in cellular viability. As another disease-modifying strategy, several efforts are undertaken to ameliorate AD-relevant symptoms by the treatment with nerve growth factor (NGF). Generally, it is known that substituted pyrimidines have modest growth-promoting effects. Here, KP544, a novel substituted pyrimidine, was characterised. This drug increased MTT reduction potential in terminally differentiated and undifferentiated PC12 cells. Furthermore, treatment with KP544 led to a reduction in Aß1-40 secretion. Thus, one may conclude that the target of KP544, GSK-3ß, represents a connecting link between the two main pathological hallmarks of AD and might thus be a very promising therapeutic target for AD.