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Pentathiepins are cyclic polysulfides that exert antiproliferative and cytotoxic activity in cancer cells, induce oxidative stress and apoptosis, and potently inhibit GPx1. These properties render this class of compounds promising candidates for the development of anticancer drugs. However, the biological effects and how they intertwine to promote high cytotoxicity have not been systematically assessed throughout a panel of cancer cell lines from distinct tissues of origin. In this thesis, six novel pentathiepins were analyzed and constitute the second generation of compounds with additional properties such as fluorescence or improved water solubility to facilitate cellular testing. All compounds underwent extensive biological evaluation in 14 human cancer cell lines. These studies included investigations of the inhibitory potential with regards to GPx1 and cell proliferation, examined the cytotoxicity in human cancer cell lines, as well as the induction of oxidative stress and DNA strand breaks. Furthermore, selected hallmarks of apoptosis, ferroptosis, and autophagy were studied. Experimental approaches regarding these cellular mechanisms included observing morphological changes, detecting phosphatidyl serine exposure and caspase activity, and quantifying cleaved PARP1 and levels of LC3B II. In addition, the analysis of the cell cycle aimed to identify aberrations or arrests in cell division.
Five of the six tested pentathiepins proved to be potent inhibitors of the GPx1, while all six exerted high cytotoxic and antiproliferative activity, although to different extents. There was a clear connection observed between the potential to provoke oxidative stress and damage to DNA in the form of single- and double-strand breaks both extra- and intracellularly. Furthermore, various experiments supported apoptosis but not ferroptosis as the mechanism of cell death in four different cell lines. In particular, the externalization of PS, the detection of activated caspases, and the cleavage of PARP1 corroborated this conclusion. Additionally, indications for autophagy were found, but more investigations are required to verify the current data. The findings of this dissertation are mainly in line with the postulated mechanism of action proposed for pentathiepins and a previous publication from our group that described their biological activity. However, the influence of modulators such as oxygen and GSH on the biological effects was ambiguous and dependent on the compound. The expression profile of the cell lines concerning GPx1 and CAT did not influence the cellular response toward the treatment, whereas the cell doubling time correlated with the cytotoxicity.
As the various pentathiepins give rise to different biological responses, modulation of the biological effects depends on the distinct chemical structures fused to the sulfur ring. This may allow for future optimization of the anticancer activity of pentathiepins. An analysis of the structure-activity relationships revealed that the piperazine scaffold was associated with superior biological activity compared to the pyrrolo-pyrazine backbone. Furthermore, substituents with electron-withdrawing properties or those providing a free electron pair, such as fluorine or morpholine, were advantageous. These findings should help design and synthesize the next generation of pentathiepins, thereby expanding the library of compounds, allowing for the further deduction of structure-activity relationships and an improved understanding of their mechanism of action.
Non-thermal atmospheric pressure plasma has drawn more and more attention to the field of wound healing research during the last two decades. It is characterized by a unique composition, which includes amongst others free radicals, ions and electrons. Furthermore, non-thermal plasma exhibits temperatures that are below those inducing thermal cell damage. Next to its well-established anti-bacterial properties, plasma can have lethal as well as stimulating effects on mammalian cells. Therefore, the medical application of non-thermal plasma on chronic wounds seems to be a promising tool to enable healing processes. However, less is known about the plasma-mediated induction of intracellular signaling pathways in human immune cells, which play a leading part in the process of wound recovery and removal of pathogens. Therefore, this thesis examined the cellular effects of a non-thermal atmospheric pressure plasma treatment on human immune cells using the argon plasma jet kinpen 09. Here, the CD4+ T helper cell line Jurkat, the monocyte cell line THP-1 as well as the corresponding primary cells were investigated. First, cell survival and apoptosis induction was assessed in response to non-thermal plasma treatment by growth curves and flow cytometric assays. On the one hand it could be shown that primary cells are more susceptible to plasma treatment than the respective cell lines. On the other hand, monocytes responded less sensitive to plasma exposure than lymphocytes. Furthermore, this thesis outlined the impact of non-thermal plasma treatment on the gene expression level of immune cells. Therefore, DNA microarray analysis was performed with the cell lines Jurkat and THP-1. It became obvious that plasma exposure modulated the expression of several genes in both cell types. Differential expression of distinct target genes was further validated by quantitative PCR in the immune cell lines. Here, elevated gene expression levels of JUN and FOS in Jurkat cells and increased transcription of JUND in THP-1 cells in response to plasma treatment were made visible. JUN, FOS and JUND are components of the transcription factor AP-1, which is involved amongst others in gene expression of IL-8 and HMOX-1. Consequently, transcriptional induction of the inflammatory cytokine IL-8 as well as the enzymes HMOX-1 and GSR was detected in plasma-treated THP-1 cells. In addition, alterations in the protein activation levels were analyzed in plasma-treated Jurkat, THP-1 cells and primary monocytes. Since some of the identified target genes are known to be associated with the MAPK pathways, the regulation of these cascades was further investigated by western blot analysis. In all investigated cell types the pro-proliferative signaling molecules ERK 1/2 and MEK 1/2 as well as the pro-apoptotic signaling proteins p38 MAPK and JNK 1/2 were activated in a plasma treatment time dependent manner. In contrast to Jurkat and primary monocytes, the anti-apoptotic HSP27 was only induced in THP-1 cells in response to plasma exposure. Moreover, modulation of cytokine production and secretion was examined in the different immune cell types and co-cultured THP-1 and HaCaT keratinocytes by ELISA or flow cytometry. While Jurkat cells showed no plasma-mediated regulation of cytokine expression, THP-1 cells revealed an increased IL-8 secretion after long plasma time duration (360 s). Additionally, the intracellular expression levels of IL-6 and IL-8 were modulated in primary monocytes by plasma exposure. While short plasma treatment caused no alteration of the number of cells expressing IL-8 an up-regulation of the intracellular IL-6 level occurred after 30 s of plasma treatment. Long plasma treatment times resulted in a significant decrease of the intracellular IL-8 and IL-6 production levels. Furthermore, co-cultured THP-1 and HaCaT cells as well as mono-cultured THP-1 and HaCaT cells were examined regarding their cytokine secretion profile. Here, cells treated with plasma (180 s) as well as LPS and plasma (180 s and LPS) were compared with untreated cells. IL-6, IL-8 and GM-CSF secretion was induced by both plasma and plasma combined with LPS treatment in mono-cultivated HaCaT cells and co-cultured cells. Though, the highest cytokine secretion levels were reached in the plasma and LPS exposed co-culture. In contrast, mono-cultivated THP-1 cells only showed an increased secretion of IL-6, IL-8 and TNFa after incubation with plasma together with LPS exposed medium. In conclusion, this study revealed for the first time the non-thermal plasma-modulated expression of numerous genes and cytokines and the activation state of various signaling cascades in human immune cells. Thus, it contributes to gain a better understanding of the immune-modulatory impacts of plasma that might promote the wound healing process.