Bedeutung der Homologen Rekombination für die Wirkung des dualen PI3K/mTOR-Inhibitors NVP-BEZ235 auf eine kombinierte Behandlung von Kopf-Hals-Tumorzellen mit Cisplatin und Bestrahlung
Die Kopf-Hals-Karzinome (HNSCC) sind weltweit betrachtet die sechst häufigste Tumorerkrankung des Menschen und weisen eine steigende Inzidenz auf. Zu den Risikofaktoren zählen neben dem Konsum von Alkohol und Nikotin die Infektion mit dem humanen Papillomavirus, häufig des Typs 16. Ätiologisch unter...
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Format: | Doctoral Thesis |
Language: | German |
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Philipps-Universität Marburg
2024
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Online Access: | PDF Full Text |
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Head and neck cancer (HNSCC) is the sixth most common tumour disease in humans worldwide and has an increasing incidence. In addition to the consumption of alcohol and nicotine, the risk factors include infection with the human papillomavirus, often type 16. Aetiologically, a distinction is therefore made between HPV-negative and HPV-positive HNSCC. The multimodal therapy for both entities is identical. In addition to surgery, radiotherapy in combination with the chemotherapeutic agent cisplatin is used as definitive or adjuvant treatment. The 5-year survival rates for HPV-positive HNSCC are around 85%, whereas they stagnate at around 50% for HPV-negative HNSCC. Due to the toxicity of cisplatin and radiotherapy, these treatments are associated with considerable side effects and restrictions in quality of life. As previous experiments in the laboratory have already shown, the PI3K/Akt/mTOR signalling pathway can be effectively inhibited by NVP-BEZ235 (BEZ235). This leads to radiation sensitisation independent of HPV status. The aim of this dissertation was to clarify the extent to which HPV status influences cellular survival when homologous recombination (HR) is inhibited by inhibiting its key protein Rad51 via siRNA transfection. This was demonstrated in three HPV-negative (Cal-33, UM-SCC-11b, UM-SCC-3) and two HPV-positive (UM-SCC-47, UD-SCC-2) cell lines. After inhibition of Rad51, the cells were treated with cisplatin, BEZ235 or radiotherapy. The different therapies were also combined with each other. Cellular survival was determined using the colony formation test. The inhibition of Rad51 was proven by Western blotting. The following observations were made: 1. plating efficiency decreases to a greater or lesser extent after inhibition of Rad51 depending on the cell line, irrespective of HPV status. 2. while the HPV-negative cell lines show a higher sensitivity to cisplatin after inhibition of Rad51, this remains identical in the HPV-positive cell lines. 3. pretreatment with BEZ235 50 nM shows no effect in combination with cisplatin in the native HPV-negative cell lines (exception UM-SCC-11b) in contrast to the HPV-positive cell lines. 4. in the case of inhibition of Rad51, the HPV-negative cell lines behave like native HPV-positive cell lines when pretreated with BEZ235 and subsequent cisplatin administration. 5 The combination of irradiation and cisplatin shows additive effects in the HPV-negative cell lines compared to irradiation alone. Inhibition of Rad51 also does not reduce cellular survival with this combination. 6 The combination of radiotherapy and BEZ235 shows an advantage in terms of lower cellular survival in HPV-negative cell lines. When Rad51 is downregulated, cellular survival decreases further with this combination. 7 The triple therapy of BEZ235, cisplatin and radiotherapy shows the lowest cellular survival in both HPV-negative and HPV-positive cell lines. This can be further reduced if the HPV-negative cell lines are subjected to downregulation of Rad51. This work thus confirms that the HPV-positive cell lines have a defective HR, while it is intact in the HPV-negative cell lines. This could explain the better prognosis of HPV-positive HNSCC. If Rad51, a key HR protein, is switched off in the HPV-negative cell lines, a similar cellular survival is observed under treatment with cisplatin, BEZ235 and/or radiotherapy as in the HPV-positive cell lines. It was also shown that BEZ235 has a pronounced radiosensitising effect and achieved the lowest cellular survival in combination with radiotherapy and cisplatin. In the future, it should be investigated whether these in vitro results of the triple therapy can also be reproduced in a clinical trial on advanced HNSCC. This could lead to a reduction in side effects by reducing the cisplatin concentration and an improvement in the prognosis of HNSCC due to the increased efficacy of radiochemotherapy.