Saudi Cultural Missions Theses & Dissertations
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Item Restricted Are extracellular vesicles mediators of chemotherapy resistance in head and neck cancer?(University of Sheffield, 2024) Alanazi, Helal; Hunt, StuartHead and neck cancer (HNC) is a term that describes malignancies that arise in areas such as the mouth and lips, larynx, pharynx, salivary glands, nose, paranasal sinus, and nasopharynx. HNC usually occurs after exposure to carcinogenic factors such as alcohol, tobacco, and human papilloma virus (HPV). Moreover, this type of cancer has a high recurrence rate at the primary site after treatment. Cisplatin is a cornerstone in the chemotherapeutic treatment of HNC, utilized for its ability to induce cancer cell death. However, resistance to cisplatin remains a significant challenge, reducing its efficacy. There is evidence that extracellular particles (EPs) such as extracellular vesicles (EVs) and vault particles mediate resistance to chemotherapy in some cancer types. However, little is known of their relative contribution, especially in HNC. Given the pivotal role of cisplatin in HNC treatment, it was hypothesized that EPs released from HNC cells exposed to cisplatin facilitate chemotherapy resistance by exporting cisplatin or by mediating intercellular communication with neighbouring cells. To test this hypothesis, MTT assay was used to determine the concentration of cisplatin required to reduce cell viability by 50% (IC50) in H357 and FaDu HNC cell lines. EV deficient (H357△HGS) and vault particle deficient (H357△MVP) were utilised to explore their contribution to cisplatin resistance. A cisplatin-resistant H357 cell line (cisplatinR) was generated by prolonged exposure to increasing concentrations of cisplatin, and putative proteins involved in resistance were quantified by western blotting. The proportion of cells undergoing apoptosis in response to cisplatin treatment was determined by flow cytometry. The concentration of EPs in cell line conditioned medium and ultracentrifugation pellets were determined by nanoparticle tracking analysis (NTA). EPs pellets were characterised by western blotting to detect EV (CD63 and TSG101) and vault particle (MVP) markers. Apoptosis was triggered in the H357 cell line following treatment with cisplatin, but not in the FaDu cell line. Treatment of H357 with cisplatin also resulted in increased release of EPs, which included EV and vault particles. Despite releasing fewer EPs in response to cisplatin treatment, the EV deficient cell line (H357△HGS) showed no significant difference in cisplatin sensitivity, whereas the vault particle deficient cell line (H357△MVP) was more resistant to cisplatin treatment. The cisplatinR 2 cell line showed increased viability and reduced apoptosis but released fewer EPs following cisplatin treatment. The findings suggest that the differential release of EPs in response to cisplatin treatment might contribute to the variability in chemotherapy resistance among HNC cell lines. Specifically, vault particles may play a more crucial role than previously understood in mediating resistance to cisplatin. This insight into the mechanistic underpinnings of cisplatin resistance in HNC cells could guide the development of novel therapeutic strategies aimed at enhancing cisplatin sensitivity by targeting the release or function of specific EPs.28 0Item Restricted Assessing the effect of cisplatin on mitochondria in monocytes and macrophages(amnah alnami, 2022-09-15) Alhassan, Alnami Amnah; Mark, PaulCisplatin is a platinum based chemotherapeutic agent which is widely used to treat a variety of solid tumours. However, the use of cisplatin is limited because of its side effects such as nephrotoxicity, neurotoxicity, ototoxicity, and peripheral neuropathy. Clinically this has become more of a problem over the past 10 years as cancer survivorship has dramatically increased. The mechanism behind how cisplatin induces toxicity is still unclear. We aim to evaluate the effects of cisplatin on mitochondrial metabolism in monocytes and macrophages. THP-1 Blue, a human acute monocytic leukaemia cell line, was used in this study. THP- 1 Blue monocytes were differentiated into macrophages using 20 ng/ml PMA. Mitochondrial activity was determined by MTT assay, and cell viability was determined using Trypan blue assay. Mitochondrial function was assessed using Mito tracker red CMROS and nano live microscopy, and the change in mitochondrial respiration was assessed using the seahorse XF Cell Mito stress test kit. NF-KB activation was measured using Quanti-Blue assay, and IL-6 was measured using an ELISA. Cisplatin dose-dependently decreased mitochondrial activity in THP-1 Blue monocytes and macrophages. Cisplatin caused mitochondrial dysfunction and induced cell death in THP-1 Blue macrophages after exposure to high dose (30 μg/ml) and even at low dose (5 μg/ml) decreased mitochondrial respiration after 24 hours. However, cisplatin treatment did not significantly affect NF-KB signaling in monocytes or macrophages. Also, IL-6 levels were undetectable in THP-1 Blue macrophages after exposure to different concentrations of cisplatin. Therefore, there is a relationship between mitochondrial dysfunction and cisplatin induced toxicity in this cell line. However, further studies are needed to investigate more on macrophages to see if cisplatin triggers inflammation.11 0