Effects of novelly synthesized nucleolipides on different tumor cell lines (HT29, HepG2, Panc-1, RenCa) with special respect to glioma cell lines (BT4Ca, GOS3, G28, G112, U251, U87) of human or other species

Today cancer is the second leading cause of death around the world. The World Health Organization predicts an increase from 9.6 million in 2018 up to 16.4 million cancer deaths in 2040. Although tumors of the brain and the nervous system are rather unusual among adults (2 %), they are the second mos...

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Main Author: Hammerbacher, Katharina
Contributors: Kinscherf, Ralf (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Published: Philipps-Universität Marburg 2020
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Summary:Today cancer is the second leading cause of death around the world. The World Health Organization predicts an increase from 9.6 million in 2018 up to 16.4 million cancer deaths in 2040. Although tumors of the brain and the nervous system are rather unusual among adults (2 %), they are the second most common diagnosed types of cancer in childhood. Gliomas and meningiomas are the most frequent types of brain tumors and account for 30 % of all tumors in the brain and central nervous system as well as for 80 % of all malignant intra-cranial tumors. Usually surgical excision is the first step in the treatment of cancer followed by radio- and chemotherapy. Due to the mainly intracranial location of gliomas, surgical interventions are extremely risky. Therefore, the use of radiation and chemotherapeutic agents become more important. 5-Fluorouracil (5-FU) is a well-known drug, usually used as chemotherapeutic agent in the standard treatment of fast proliferating cancer cells. Unfortunately, it carries along a wide range of unpleasant side effects in multiple organ systems. To improve the passage through the blood-brain-barrier and the cell membrane penetration thereby gaining a higher efficiency, our cooperation partners Prof. Dr. Rosemeyer and his colleagues from the Institute of Chemistry and Materials of the University of Osnabrück synthesized more than 120 novel nucleolipids. These molecules have been designed based on the 5-FU derivative 5-Fluorouridine (5-FUrd) or the nucleosides adenosine, cladribin, formycin, guanosine, inosine and others. The derivatives are modified at several positions and have different additional chemical side groups to mainly influence the hydrophilicity and lipophilicity and thus, to consequently improve the permeability across the blood-brain-barrier as well as the uptake into the cells. At first we screened the impact of 5-FUrd and the 120 novelly synthesized derivatives on the viability of rat BT4Ca and human GOS3 glioma cells as well as differentiated human macro-phages (THP1). Five derivatives (S.18, 19, 38, 98 and 101), which show low cytotoxic effects on the macrophages and are effective against both, the rat BT4Ca and the human GOS3 glioma cells, were selected to be further analyzed. Although we screened 120 derivatives consisting of purines and pyrimidines, it is remarkable that all five selected, most effective derivatives (S.18, 19, 38, 98 and 101) belong to the group of pyrimidines. Comparing the chemical structure of the selected derivatives, the position of the attached farnesyl chain seems to be of high importance for the effectiveness of the most efficient derivative (S.98) against rat as well as human glioma cell lines. We were able to show that the selected derivatives, derived out of the nucleosides adenosine (S.38), formycin (S.98) and inosine (S.18, 19 and 101) are able to keep up with or even overcome the cytostatic/cytotoxic effects of the well-known chemotherapeutic agent 5-FU on further human glioma cell lines (G28, G112, U251 and U87) and several other tumor entities (HT29, HepG2, Panc-1 and RenCa). Certainly, these effects depend on the applied derivative as well as its concentration and vary among the specific cell type analyzed. Furthermore, using the rat BT4Ca glioma cells as a model of fast proliferating glioblastomas, we studied several intracellular mechanisms, possibly responsible for the examined cytotoxic effects of the selected derivatives (S.18, 19, 38, 98 and 101). Our results indicate that the treatment of the rat BT4Ca glioma cells with each derivative lead to a concentration-dependent increase of the apoptotic cell rate. At concentrations 25 and 50 µM each of the selected derivatives led to a distinct activation of the caspase 3, confirming the induction of apoptosis. We also observed an increase of the necrotic cell rate with ascending concentration (12.5, 25 and 50 µM) of each derivative presumably due to secondary necrosis. Beside the loss of cell mass due to apoptosis, we detected lower levels of PCNA after the treatment with any selected derivative (12.5 and 25 µM) indicating a reduced cell proliferation of the rat BT4Ca glioma cells. In contrast, the treatment of the rat BT4Ca glioma cells with 5-FUrd did not affect the amount of PCNA. Thus, at our experimental conditions 5-FUrd showed no impact on the proliferation rate consistent with its cytostatic effects. Moreover, we determined a tremendous accumulation of ROS per cell after the treatment with each derivative (50 µM). Especially the treatment with the derivatives S.18, 38 and 98 caused high oxidative stress in the rat BT4Ca glioma cells. But regarding the ratio of rGSH/GSSG only the derivative S.98 showed an explicit effect on the glutathione antioxidant system by lowering the rGSH/GSSG ratio. Nevertheless, the activation of NFκB, through the translocation of the subunit p65 from the cytoplasm to the nucleus, was not triggered by the treatment with any selected derivative or 5-FUrd. For a validation of the effects of the derivatives on the migratory capacity of the rat BT4Ca glioma cells during the treatment, further experiments need to be done with a more accurate method. The results indicate that our five selected derivatives S.18, 19, 38, 98 and 101 bear fundamen-tal attributes, which are beneficial for the treatment of malignant cancer cells. These proper-ties and the broad range of cytotoxicity as well as the inhibition of the cell proliferation make the five derivatives, especially S.19 and S.98, highly interesting as prospects against several tumor entities for the use as possible novel drugs with a high potential as chemotherapeutic agent.
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