Refine
Year of publication
- 2020 (235) (remove)
Document Type
- Doctoral Thesis (235) (remove)
Has Fulltext
- yes (235)
Is part of the Bibliography
- no (235)
Keywords
- Knochenersatzmaterial (2)
- Sprache (2)
- 3-alkylphenols (1)
- 3D-Druck (1)
- 6-methylsalicylic acid synthase (1)
- 9-HODE (1)
- ADHS (1)
- ALL (1)
- AML (1)
- Absorption modeling (1)
Institute
Nematophilic bacteria as a source of novel macrocyclised antimicrobial non-ribosomal peptides
(2020)
A solution to ineffective clinical antimicrobials is the discovery of new ones from under-explored sources such as macrocyclic non-ribosomal peptides (NRP) from nematophilic bacteria. In this dissertation an antimicrobial discovery process –from soil sample to inhibitory peptide– is demonstrated through investigations on six nematophilic bacteria: Xenorhabdus griffiniae XN45, X. griffiniae VH1, Xenorhabdus sp. nov. BG5, Xenorhabdus sp. nov. BMMCB, X. ishibashii and Photorhabdus temperata. To demonstrate the first step of bacterium isolation and species delineation, endosymbionts were isolated from Steinernema sp. strains BG5 and VH1 that were isolated directly from soil samples in Western Kenya. After genome sequencing and assembly of novel Xenorhabdus isolates VH1 and BG5, species delineation was done via three overall genome relatedness indices. VH1 was identified as X. griffiniae VH1, BG5 as Xenorhabdus sp. nov. BG5 and X. griffiniae BMMCB was emended to Xenorhabdus sp. nov. BMMCB. The nematode host of X. griffiniae XN45, Steinernema sp. scarpo was highlighted as a putative novel species. To demonstrate the second step of genome mining and macrocyclic non-ribosomal peptide structure elucidation, chemosynthesis and biosynthesis, the non-ribosomal peptide whose production is encoded by the ishA-B genes in X. ishibashii was investigated. Through a combination of refactoring the ishA-B operon by a promoter exchange mechanism, isotope labelling experiments, high resolution tandem mass spectrometry analysis, bioinformatic protein domain analysis and chemoinformatic comparisons of actual to hypothetical mass spectrometry spectra, the structures of Ishipeptides were elucidated and confirmed by chemical synthesis. Ishipeptide A was a branch cyclic depsidodecapeptide macrocyclised via an ester bond between serine and the terminal glutamate. It chemosynthesis route was via a late stage macrolactamation and linearised Ishipeptide B was synthesised via solid phase iterative synthesis. Ishipeptides were not N-terminally acylated despite being biosynthesised from the IshA protein that had a C-starter domain. It was highlighted that more than restoration of the histidine active site of this domain is required to restore N-terminal acylation activity.
To demonstrate the final step of determination of antimicrobial activity, minimum inhibitory concentrations of Ishipeptides and Photoditritide from Photorhabdus temperata against fungi and bacteria were determined. None were antifungal while only the macrocyclic compounds were inhibitory, with Ishipeptide A inhibitory to Gram-positive bacteria at 37 µM. The cationic Photoditritide, a cyclic hexapeptide macrocyclised via a lactam bond between homoarginine and tryptophan, was 12 times more inhibitory (3.0 µM), even more effective than a current clinical compound, Ampicillin (4.2 µM). For both, macrocyclisation was hypothesised to contribute to antimicrobial activity. Ultimately, this dissertation demonstrated not only nematophilic bacteria as a source of novel macrocyclic antimicrobial non-ribosomal peptides but also a process of antimicrobial discovery–from soil sample to inhibitory peptide– from these useful bacteria genera. This is significant for the fight against antimicrobial resistance.
Although immune checkpoint inhibitors such as anti-PD-1 antibodies have shown remarkable clinical success in many different tumor types, the proportion of patients benefiting from this treatment option remains low. Therefore, there is a need to sensitize tumors for immune checkpoint blockade. In this study two approaches were tested, a chemoimmunotherapy approach combining PD-1 checkpoint blockade with doxorubicin (DOX) chemotherapy, and ablation of the sphingosine-1-phosphate (S1P) receptor (S1PR4) based on the following rationale. Chemotherapy was shown to induce immune paralysis which contributes to tumor relapse, while PD-1 signaling was shown to facilitate the acquisition of chemoresistance. Thus, combinatorial chemoimmunotherapy is expected to be beneficial by maintaining or even activating anti-tumor immunity during chemotherapy. S1PR4 is an immune cell specific receptor, whose ablation slowed tumor progression by activating anti-tumor immunity in a mouse model that was previously insensitive to anti-PD-1 monotherapy. This suggested that S1PR4 ablation might pre-activate immunity to sensitize for anti-PD-1 therapy.
To test these combinatorial approaches, two tumor mouse models were employed, namely the MC38 murine adenocarcinoma model as well as the transgenic polyoma middle T oncogene (PyMT) breast cancer model. In the MC38 model, a mild synergistic effect of PD-1 immune checkpoint blockade and S1PR4 ablation was observed, indicated by improved tumor progression and survival as compared to the WT control, and an increased number of tumor-free mice compared to anti-PD-1 therapy alone in WT mice. These observations correlated with an enhanced natural killer (NK) cell infiltrate and increased CXCL9 and CXCL10 production in anti-PD-1 treated S1PR4 KO tumors. As noted before, the PyMT model was largely resistant to anti-PD-1 monotherapy in a therapeutic setting. S1PR4 ablation alone showed significant tumor reduction that was not further enhanced by anti-PD-1 treatment. The same was observed when chemotherapy with DOX was added, where WT tumors relapsed, while S1PR4 KO tumor did not. Addition of anti-PD-1 did only mildly increase tumor control in S1PR4 KO mice, indicating that S1PR4 KO per se very efficiently re-activated anti-tumor immunity. Since S1PR4 KO induces type I 12 interferon (IFN-1) over-production in S1PR4 KO PyMT tumors, a link between high IFN-1 levels and tumor immunity was tested by using mice deficient in the IFN-1 receptor (IFNAR1). Unexpectedly, DOX chemotherapy was most efficient in mice with IFNAR ablation only as compared to WT, S1PR4 KO or S1PR4 and IFNAR1 double KO mice, although deficiency in IFNAR signaling is predominantly regarded as tumor promoting. The underlying mechanisms need to be tested in future studies. Interestingly, chemoimmunotherapy in WT mice prevented tumor relapse to a similar extent than S1PR4 KO and was superior to chemotherapy or immune checkpoint blockade alone. To investigate mechanisms of chemoimmunotherapy success compared to monotherapy, whole transcriptome analysis was used, which identified a set of genes that were upregulated specifically upon chemoimmunotherapy. This gene signature and, more specifically, a condensed four-gene signature predicted favorable survival of human mammary carcinoma patients in the METABRIC cohort.
Moreover, PyMT tumors treated with chemoimmunotherapy contained higher levels of cytotoxic lymphocytes, particularly NK cells. Gene set enrichment analysis and ELISA measurements revealed increased IL-27 production and signaling in PyMT tumors upon chemoimmunotherapy. Moreover, IL-27 improved NK cell cytotoxicity against PyMT cells in vitro. These data supported recent clinical observations indicating a benefit of chemoimmunotherapy compared to monotherapy in breast cancer and suggested potential underlying mechanisms.
Taken together the present work revealed new strategies to reactivate tumor immunity leading to improved chemotherapy response, namely a combination with immune checkpoint blockade and ablation of S1PR4, which activated different lymphocyte compartments within tumors.
The interleukin (IL)-1 family has been described for its numerous involvement in the regulation of inflammatory processes. Certain members are able to induce inflammation, whereas others have the capacity to inhibit inflammation. The newly discovered IL-1 family member IL-38 shows interesting and innovative properties. While most of these cytokines are pro-inflammatory mediators, IL-38 appears to enter the smaller circle of anti-inflammatory mediators. As a pattern, IL-38 appears to suppress IL-17-driven chronic or auto-inflammation by working as receptor antagonist. These properties, as well as its beneficial effects in models of inflammatory and autoimmune diseases suggest the possibility of IL-38-based therapies. Nevertheless, its role in the resolution of acute inflammation, thereby preventing chronic inflammation, remains unclear.
The first part of my thesis elucidated the role of IL-38 in the resolution of inflammation. I found that the complete absence of IL-38 in IL-38 KO mice leads to a delayed resolution of inflammation in the zymosan-induced peritonitis mouse model, compared to WT mice. This was marked by a persistent neutrophilia and a lower production of pro-resolving mediators during the resolution phase, such as TGFβ1 production from macrophages following efferocytosis of apoptotic cells. Reduced TGFβ1 production from macrophages coincided with reduced levels of regulatory T cells (Tregs), which are known to promote the resolution of inflammation. Unexpectedly, the TGFβ1 production capacity of macrophages did not influence the induction of Tregs from naïve T cells. Rather, IL-38 KO mice had an accumulation of Tregs in the thymus compared to WT mice. This was caused by an impairment of CD62L expression at the surface of Tregs, which is required for Tregs migration outside of the thymus. Higher Treg numbers in the thymus correlated with lower level of Tregs in peripheral lymphoid organs. Importantly, CD62L expression at the surface of IL-38 KO Tregs in the thymus was restored by injecting IL-38 i.p. for 24h. These data indicate a potential key function of IL-38 in the regulation of Treg migration, which is triggered in many cases of autoimmunity.
The second part of my thesis was to study the role of IL-38 in experimental autoimmune encephalomyelitis (EAE) development, given that EAE is IL-17-dependent. Unexpectedly, IL-38-deficient mice showed strongly reduced clinical scores and histological markers of EAE. This came with reduced inflammatory cell infiltrates, as well as reduced expression of inflammatory markers in the spinal cord. IL-38 mRNA was detected in the spinal cord, mainly by resident and infiltrated phagocytes, but also by other cells, such as ependymal cells. IL-38 was upregulated upon pro-inflammatory stimulation of bone marrow-derived macrophages, and its presence was necessary for a complete activation of inflammatory macrophages. My data suggest an alternative cell-intrinsic role of IL-38 in macrophages to promote inflammation in the central nervous system.
In the last part of my thesis, I initiated a project on the function of IL-38 in B cell physiology and antibody production, given the fact that IL-38 is expressed by B cells. I generated preliminary data showing that the absence of IL-38 in mice decreased antibody production. Furthermore, I showed that IL-38 is particularly expressed by plasma cells in human tonsils. This project remains open and further studies will be conducted to investigate how IL-38 regulates antibody production, both in physiological and autoimmune settings. Understanding the role of IL-38 in autoantibody production could lead to original and innovative therapy for patients suffering from auto-inflammatory disease.
In summary, the different projects of my thesis provide evidence that the pro-resolving function of IL-38 may be indirectly linked to the retention of Tregs in the thymus. Moreover, a possible intracellular role of IL-38 within macrophages was described showing opposite properties in the regulation of inflammation. This function could be causatively involved in EAE development. However, further studies remain to be done to find the mechanism of action by which IL-38 regulates Tregs egression and how it influences the EAE development. Complete understanding of the IL-38 biology and differentiation between its extra- vs potential intracellular functions could make it a promising therapeutic target for chronic inflammatory or autoimmune diseases.
The compound class of the fabclavines was described as secondary or specialized metabolites (SM) for Xenorhabdus budapestensis and X. szentirmaii. Their corresponding structure was elucidated by NMR and further derivatives could be identified in both strains. Biochemically, fabclavines are hybrid SMs derived from two non-ribosomal-peptide-synthetases (NRPS), one type I polyketide-synthase (PKS) and polyunsaturated fatty acid (PUFA) synthases. In detail, a hexapeptide is connected via partially reduced polyketide units to an unsual polyamine. Structurally, they are related to the (pre-)zeamines, described for Serratia plymuthica and Dickeya zeae. Fabclavines exhibit a broad-spectrum bioactivity against a variety of different organisms like Grampositive and Gram-negative bacteria, fungi, protozoa but also against eukaryotic celllines.
In this work, the fabclavine biosynthesis was elucidated and assigned to two independently working assembly lines. The NRPS-PKS-pathway is initiated by the first NRPS FclI via generation of a tetrapeptide, which is elongated by the second NRPS FclJ, leading to a hexapeptide. Alternatively, FclJ can also act as direct start of the biosynthesis, resulting in the final formation of shortened fabclavine derivatives with a diinstead of a hexapeptide. In both cases, the peptide moiety is transferred to the iterative type I PKS FclK, leading to an elongation with partially reduced polyketide units. The resulting NRPS-PKS-intermediate is still enzyme-bound. The PUFA-homologues FclC, FclD and FclE in combination with FclF, FclG and FclH belong to the polyamine-forming pathway. Briefly, repeating decarboxylative Claisen thioester condensation reactions of acyl-coenzym A building blocks lead to the generation of an acyl chain in a PKS- or fatty acid biosynthesis-like manner. The corresponding β-keto-groups are either completely reduced or transaminated in a specific and repetitive way, resulting in the concatenation of so-called amine-units. The final β-keto-group is reduced to a hydroxy-group and the intermediate is reductively released by the thioester reductase FclG. A subsequent transamination step leads to the final polyamine. The NRPS-PKS- as well as the polyamine-pathway are connected by FclL. This condensation domain-like protein catalyzes the condensation of the polyamine with the NRPS-PKS-part, which results in the release of the final fabclavine. The results are described in detail in the first publication (first author).
Fabclavine biosynthesis gene cluster (BGC) are widely spread among the genus Xenorhabdus and Photorhabdus. In Xenorhabdus strains a high degree of conservation regarding the BGC synteny as well as the identity of single proteins can be observed. However, Photorhabdus strains harbor only the PUFA-homologues. While in Photorhabdus no product could be detected, our analysis revealed that the Xenorhabdus strains produce a large chemical diversity of different derivatives. Briefly, the general backbone of the fabclavines is conserved and only four chemical moieties are variable: The second and last amino acids of the NRPS-part, the number of incorporated polyketide units as well as the number of amine units in the polyamine. In combination with the elucidated biosynthesis, these variables could be assigned to single biosynthesis components as diversity mechanisms. Together with the 10 already described derivatives, a total of 32 derivatives could be detected. Interestingly, except for taxonomic closely related strains, all analyzed strains produce their own set of derivatives. Finally, we could confirm that the fabclavines are the major bioactive compound class in the analyzed strains under laboratory conditions. The results are described in detail in the second publication (first author).
Together with our collaboration partner Prof. Selcuk Hazir a potent bioactivity against Enterococcus faecalis, which is associated with endodontic infections, could be contributed to X. cabanillasii. Here, we could confirm that this bioactivity can be assigned to the fabclavines. The results are described in detail in the third publication(co-author).
Among the genus Xenorhabdus, X. bovienii represents an exception as its NRPS and PKS genes of the fabclavine BGC are missing or truncated, resulting in the exclusive production of polyamines. Furthermore, its PUFA-homologue FclC harbors an additional dehydratase (DH) domain. Upon extensive analysis a yet unknown deoxy-polyamine was identified and assigned to this additional domain. Finally, the DH domain was transferred into other polyamine pathways. Regardless of an in cis or in trans integration, the chimeric pathways produced deoxy-derivatives of its naturally occurring polyamines, suggesting that this represents another diversification mechanism. The results are described in detail in the attached manuscript (first author).
Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1−GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.
Monoterpenes and their monoterpenoid derivatives form a subclass of terpene(oid)s. They are widely used in medicines/pharmaceuticals, as flavor and fragrance compounds, or in agriculture and are also considered as future biofuels. However, for many of these substances, the extraction from natural sources poses challenges such as occurring at low concentrations in their raw material or because the natural sources are diminishing. Furthermore, many of the structurally more complex terpenoids cannot be chemically synthesized in an economic way. Therefore, microbial production provides an attractive alternative, taking advantage of the often distinct regio- and stereoselectivity of enzymatic reactions. However, monoterpenes and monoterpenoids are challenging products for industrial biotechnology processes due to their pronounced cytotoxicity, which complicates the production in microorganisms compared to longer-chain terpenes (sesquiterpenes, diterpenes, etc.).
The aim of this thesis was to generate a biotechnological complement to fossil-resources-based chemical processes for industrial monoterpenoid production. Therefore, a starting point for the further development of a microbial cell factory based on the microbe Pseudomonas putida KT2440 was aimed to be created. This production organism should be able to conduct a whole- cell biocatalysis to selectively oxyfunctionalize monoterpene hydrocarbons using renewable industrial by-products and waste streams as raw material for monoterpenoid production (Figure 1). As a model substance, the production of (-)-menthol should be addressed due to its industrial significance. (-)-Menthol is one of the world’s most widely-used flavor and fragrance compounds by volume as well as a medical component, having an annual production volume of over 30,000 tons. An approach for (-)-menthol production from renewable resources could be a biotechnological(-chemical) two-step conversion (Figure 1), starting from (+)-limonene, a by-product of the citrus fruit processing industry.
The thesis project was divided into three parts. In the first part, enzymes (limonene-3- hydroxylases) were to be identified that can convert (+)-limonene into the precursor of (-)-menthol, (+)-trans-isopiperitenol. To counteract product toxicity, in the second part, the tolerance of the intended production organism P. putida KT2440 towards monoterpenes and their monoterpenoid derivatives should be increased. Finally, in the third part, the identified hydroxylase enzymes would be expressed in the improved P. putida KT2440 strain to create a whole-cell biocatalyst for the first reaction step of a two-step (-)-menthol production, starting from (+)-limonene.
To achieve these objectives, different genetic/molecular biology and analytical methods were applied. In this way, two cytochrome P450 monooxygenase enzymes from the fungi Aureobasidium pullulans and Hormonema carpetanum could be identified and functionally expressed in Pichia pastoris, which can catalyze the intended hydroxylation reaction on (+) limonene with high stereo- and regioselectivity. A further characterization of the enzyme from A. pullulans showed that apart from (+) limonene the protein can also hydroxylate ( ) limonene, - and -pinene, as well as 3-carene.
Furthermore, within this thesis, mechanisms of microbial monoterpenoid resistance of P. putida could be identified. It was shown that the different monoterpenes and monoterpenoids tested have very different toxicity levels and that mainly the Ttg efflux pumps of P. putida GS1 are responsible for the tolerance to many of these compounds. Based on these results, a P. putida KT2440 strain with increased resistance to various monoterpenoids, including isopiperitenol, could then be generated, which can be used as a host organism for the further development of monoterpenoid-producing cell factories.
While within the scope of this work the heterologous expression of the fungal gene in prokaryotic cells in a functional form could not be realized despite different approaches, the identified enzymes, the monoterpenoid-tolerant P. putida strain and a plasmid developed for heterologous gene expression in P. putida provide a starting point for the further design of a microbial cell factory for biotechnological monoterpenoid production.
Reliable and efficient recording of the error-related negativity with a speeded Eriksen Flanker task
(2020)
There is accumulating evidence that the error-related negativity (ERN), an event-related potential elicited after erroneous actions, is altered in different psychiatric disorders and may help to guide treatment options. Thus, the ERN is a promising candidate as a psychiatric biomarker. Basic methodological requirements for a biomarker are standardized and reliable measurements. Additional psychiatry specific requirements are time efficiency and patient-friendliness.
The aim of the present study is to establish ERN acquisition in a reliable, time-efficient and patient-friendly way for use in clinical practice.
Healthy subjects (N=27) performed a modified Eriksen Flanker Task with adaptive reaction time window and only incongruent stimuli that maximizes the number of errors. All participants were tested for mental health by the Mini International Neuropsychiatric Interview (M.I.N.I.). The first N=12 subjects were part of a pilot study and further N=14 subjects were included for analysis (one subject was excluded due to technical problems). In a test-retest design with two sessions separated by 28 days the reliability of the ERN has been assessed. To ensure external validity, we aimed to replicate previously reported correlation patterns of ERN amplitude with (1) number of errors and (2) negative affect. State affect of each subject was measured by the Positive and Negative Affect Schedule. In order to optimize the clinical use of the task, we determined to which extent the task can be shortened while keeping reliability >0.80.
We found excellent reliability of the ERN (intraclass correlation coefficient =0.806-0.947) and replicated specific correlation patterns (ERN amplitude with relative number of errors: r=0.394; p=0.082; ERN amplitude with negative affect: r=-0.583, p=0.014). The task can be shortened to a patient-friendly and clinically feasible length of only 8 minutes keeping reliability >0.80.
To conclude, the present modified task provides reliable and efficient recording of the ERN, facilitating its use as a psychiatric biomarker.
This thesis primarily investigates an (hitherto unnoticed) agreement alternation between Romance and Germanic in D>N>&>N constructions (e.g. “these walls and ceiling”). While Romance exhibits left conjunct agreement, Germanic shows morphologically resolved agreement on the determiner, i.e. the phi-feature mismatching conjuncts can only be licensed if a syncretic form is available. To handle these data the author suggests a theory in which coordination is syntactically and morphologically unspecified and multiple agree is a general option. Infelicitous derivations are ruled out by interface conditions on the semantic and the phonological well formedness. The complete results of the corpus research conducted to deliver a sound empirical basis for the phenomena investigated in this thesis can be found in the appendix.
Evidence is increasingly pointing towards a significant global decline in biodiversity. The drivers of this decline are numerous, including habitat change and overexploitation, rapid deforestation, pollution, exotic species and disease, and finally climate change as an emerging driver of biodiversity change (Nakamura, et al., 2013; Hancocks, 2001; Pereira, Navarro & Martins, 2012). Raising public awareness of the need to conserve biological diversity is essential to safeguard the richness of life forms all over the world (Lindemann-Matthies, 2002). In this regard, institutions such as science museums, zoos and aquariums have the potential to play an important role (Rennie & Stocklmayer, 2003). Especially, zoos can provide a productive learning environment (Miles & Tout, 1992), facilitating the promotion of public conservation awareness and the adoption of pro-environmental behaviours that would reduce negative human impacts on biodiversity (Barongi, et al., 2015).
Based on these concepts, my study contributes to the developing field of visitor studies. Taking as reference non-zoo visitors and zoo visitors, I have focused on reviewing some aspects of conservation education, such as people's awareness of conservation, people's interest in animals and people's feelings towards animals and attitudes towards zoos. The study identified differences between non-regular and regular zoo visitors in interests in animals, as well as visitor attitudes towards conservation issues and zoos. Therefore, the present study indicated that positive emotional reactions and, in particular, a perceived sense of connection to the animal were linked and depended on the frequency of zoo visits. It was as well remarkable, that conservation awareness was influenced by the interest in animals, the interest in visiting zoos, the attitudes towards these institutions, and the age and the country of origin. All these variables had a greater effect in the conservation consciousness of the participants. Additionally interestingly, the main reason for visiting zoos in every country was to learn something about animals. This highlights the educational role of zoos and broadly supports the idea that people want to visit zoos to learn something about animals, in turn facilitating pro-conservation learning and changes in attitude. They are uniquely positioned to interact with visitors, communities, and society and to contribute by providing an informative and entertaining environment. Visiting zoos could led to contribute to promoting animal connectedness and interest in species.
Das Hepatozelluläre Karzinom (HCC) ist die sechsthäufigste Krebsart mit der zweithäufigsten krebsbedingten Letalität. Sorafenib ist bereits seit über 10 Jahren die einzige verfügbare und zugelassene systemische Chemotherapie. Allerdings zeigen Patienten oft eine Resistenz gegenüber Sorafenib.
In zahlreichen Krebsarten konnte bereits gezeigt werden, dass Sphingolipide bei der Tumorentwicklung und Chemoresistenz eine wichtige Rolle spielen. Sphingolipide sind bioaktive Lipidmoleküle, welche unteranderem für die Beeinflussung verschiedener Signalwege intra- und extrazellulär verantwortlich sind. So konnte gezeigt werden, dass das Verhältnis zwischen Sphingosin-1-Phosphat (S1P) und Ceramiden eine wichtige Rolle für das Überleben von Zellen spielt, wobei eine Verschiebung des Verhältnisses zugunsten des S1P meist eine proliferative Wirkung auf Zellen hat. Für die Phosphorylierung des Sphingosins zu S1P sind die zwei Enzyme Sphingosinkinase 1 und 2 (SPHK1/2) verantwortlich.
Es gibt bereits Studien, die nachweisen konnten, dass diese Enzyme gerade in Tumorzellen verstärkt exprimiert werden. Auf der anderen Seite kann eine verstärkte Bildung von Ceramiden die Apoptose der Tumorzellen verstärken. Daher ist es nicht verwunderlich, dass der Sphingolipid-Stoffwechsel einen interessanten Angriffspunkt für die Krebstherapie darstellt. Aus diesem Grund sollte in der vorliegenden Arbeit zunächst untersucht werden, ob Sorafenib einen Einfluss auf den Sphingolipid Stoffwechsel hat. Weiter sollte untersucht werden, ob die Beeinflussung des Sphingolipid-Stoffwechsels die Effekte von Sorafenib potenzieren könnte.
Es konnte zunächst gezeigt werden, dass Sorafenib die mRNA-Expression verschiedener Enzyme, die das Verhältnis zwischen Ceramiden und S1P regulieren, beeinflussen kann. Es wurde weiter untersucht, wie sich der Ceramidsynthase Inhibitor FB1 auf die Proliferation und die Induktion der Apoptose auswirkt. Dies wurde ebenfalls mit SLP (SPHK1 Inhibitor), SLM (SPHK2 Inhibitor) und SKI II, einem unspezifischen Inhibitor beider Sphingosinkinasen, untersucht. Es wurden weiter die Einflüsse aller verwendeten Substanzen auf die Bildung verschiedener Sphingolipide untersucht.
Hierbei konnte gezeigt werden, dass Sorafenib in der Lage ist, die Proliferation der Zellen zu hemmen und die Apoptose-Induktion zu fördern. Weiter führte Sorafenib zu einer Akkumulation der Dihydroceramide. Was wiederrum weder mit SKI II, FB1; SLP noch SLM beobachtet werden konnte. Der signifikante Anstieg der Dihydroceramid-Konzentrationen konnte mit dem durch Sorafenib induzierten oxidativen Stress in Verbindung gebracht werden.
SLP und SLM waren nicht in der Lage, die Effekte von Sorafenib auf die Proliferation zu potenzieren. Die Kombination von Sorafenib mit SLP oder SLM führte jedoch in den Huh7.5 Zellen zu einer drastischen Reduktion des S1P-Spiegel.
FB1 und SKI II führten zu einer stärkeren Hemmung der Proliferation als Sorafenib. Wobei gezeigt werden konnte, dass beide Substanzen die Ceramid-Spiegel tendenziell eher vermindern und die S1P-Spiegel erhöhen. Durch die Stimulation mit FB1 kam es sogar zu einer signifikanten Erhöhung der S1P-Spiegel. Es scheint, dass der Einfluss von FB1 und SKI II auf die Proliferation der Zellen unabhängig vom Sphingolipid-Stoffwechsel ist. Diese scheinen eher über andere Mechanismen zu wirken. Es könnte von Interesse sein, gerade diese Signalwege von SKI II im HCC weiter zu untersuchen, da SKI II bereits in Mausmodellen anderer Krebsarten vielversprechende Ergebnisse zeigte.