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Chemically Modified Adenosine Derivatives and Their Potential Therapeutic Benefits in the Treatment of Glioblastoma, Compared to the Chemotherapeutical Drug Temozolomide
Exploring and developing novel therapeutic approaches and drugs for treating various cancers represents a central, globally relevant research area. The discovery of novel nucleolipids has emerged as a promising therapeutic option, the potential of which should be comprehensively investigated and exp...
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| Format: | Doctoral Thesis |
| Language: | English |
| Published: |
Philipps-Universität Marburg
2024
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| Online Access: | PDF Full Text |
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| Summary: | Exploring and developing novel therapeutic approaches and drugs for treating various cancers represents a central, globally relevant research area. The discovery of novel nucleolipids has emerged as a promising therapeutic option, the potential of which should be comprehensively investigated and exploited further.
Within the scope of this research project, 27 nucleolipids were synthesized based on the nucleoside adenosine, including derivatives with extended and short-chain alkyl groups in various configurations, such as symmetrical, asymmetrical, and cyclic structures. Additionally, the nucleobase was modified by introducing a third ring to 1,N6-ethenoadenosine. In the context of this dissertation the synthesized nucleolipid derivatives were extensively characterized using 1H and 13C NMR spectroscopy, HR-ESI-MS, UV-Vis and fluorescence spectroscopy, and thin-layer chromatography (TLC) to determine structure, conformation, and mass.
The biophysical properties of the derivatives were analyzed with a focus on their ability to traverse the blood-brain barrier. Experimental tests indicated that compounds with an alkyl chain length of 11-19 can penetrate this barrier. Their efficacy was tested on rat glioma cells (BT4Ca) and human glioma cells (GOS-3) and compared with 5-FUrd. Initial side effect assessments were conducted on THP-1 macrophages. Further tests were performed on human glioblastoma cell lines U87, U251, G28, and G112 and the murine glioblastoma cell line GL-261, comparing the most promising 18 derivatives with the standard therapeutic agent TMZ.
The 1,N6-ethenoadenosine derivatives, particularly εNL_5.7.0.0, εNL_5.72.0.0, εNL_5.cycl7.0.0, εNL_5.83.0.0, and εNL_5.9.0.0 exhibited higher cytotoxicity than the adenosine derivatives. The derivatives demonstrated cytotoxicity against glioblastoma cells at concentrations of 6.25 µM and 12.5 µM, respectively. In contrast, they only became cytotoxic to THP-1 macrophages at a concentration of 25 µM. Additionally, the derivatives reduced cell viability more effectively than 5-FUrd and TMZ. The growth behavior of treated glioblastoma cells was examined over 336 hours. Cell viability remained stagnant at high dosages, while at lower dosages, it decreased. The cell count decreased significantly after re-treatment at 240 hours without indicating resistant clones.
In the context of a detailed investigation into the mechanism of action of the nucleolipids, a multitude of biochemical and cell biological assays were employed. Specific staining techniques were used to characterize the type of cell death mechanisms. Stains using YoPro™-1 and PI were utilized to detect apoptosis and necrosis. Annexin V staining was also applied to identify and quantify apoptotic cells precisely. A central aspect of this investigation was analyzing the mitochondrial membrane potential, a crucial parameter for mitochondrial integrity and function, using JC-10 staining. These analyses showed an initially increased activity, which was subsequently accompanied by depolarization. These observations strongly suggest the involvement of mitochondria in the apoptotic processes, indicating mitochondrial apoptosis is closely linked with the intrinsic apoptotic pathway.
In addition to the cell biological assays, Western blot analyses were conducted to quantify the expression of specific proteins. The proteins RIPK3, BAX, BID, p62, and LC3 were the focus of the investigations. These analyses revealed a significant increase in the expression of BAX and LC3-II. Furthermore, the results indicated an enhanced formation of autophagosomes, suggesting the involvement of autophagic processes in the mechanism of action of the nucleolipids.
The localization of the 1,N6-ethenoadenosine derivatives within the cell was determined using confocal laser scanning microscopy. Nuclear and plasma membrane stains served as reference points. The analysis revealed that the compounds predominantly reside near the cell nucleus in the cytoplasm, providing valuable insights into their cellular distribution and potential target structures. Electron microscopy studies were also conducted to understand cellular changes comprehensively. These high-resolution analyses validated the outcomes of the preceding investigations, such as the induction of apoptosis and autophagy.
The collected data from the various experimental approaches suggest that induced apoptosis and the involvement of autophagic processes are essential underlying mechanisms of action of the nucleolipid derivatives.
Due to their cytotoxic properties, the six selected derivatives, particularly εNL_5.9.0.0, exhibit promising potential as novel agents in chemotherapy, surpassing the efficacy of the standard therapeutic agent TMZ. It is worth noting that the efficiency of these derivatives/substances is planned to be further investigated through animal experiments using tumor-bearing mice, e.g., in glioblastoma, colon, and pancreatic cancer models, and compared to control substances like 5-FU and TMZ. |
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| DOI: | 10.17192/z2024.0285 |