1. UKM Learning and Research Repository
2. PHD Thesis / Tesis PHD
3. Faculty of Pharmacy / Fakulti Farmasi

Title:	Evaluation of influence of mithochondrial DNA alterations on cisplatin responsiveness and identification of bioactive compounds synegizing cisplatin sensitivity in oral squamous cell carcinoma
Supervisor:	Ng Pei Yuen, Dr.
Keywords:	DNA Mitochondrial Carcinoma Squamous Cell Cisplatin
Issue Date:	17-Aug-2021
Description:	Cisplatin is the chemotherapeutic agent of choice for oral squamous cell carcinoma (OSCC). Nevertheless, the differential sensitivity to cisplatin due to intrinsic or acquired resistance remains as an obstacle to its chemotherapeutic efficacy. Recently, mitochondrial DNA (mtDNA) alterations and cancer stem cells (CSCs) model have been reported as the basis of treatment resistance and recurrence. However, the role of modifications in mtDNA, which encodes mitochondrial oxidative phosphorylation complexes, in OSCC has not been comprehensively studied. The CSCs also are not being targeted in the current chemotherapy. Thus, this study aimed to evaluate the role of mtDNA alterations on cisplatin responsiveness in OSCC, and to identify bioactive compounds against a cisplatin-resistant CSCs model derived from OSCC through a high-throughput screening (HTS). To examine intrinsic and acquired cisplatin-resistant cell models, CSCs model or tumour spheres with stem cell-like features and cisplatin-resistant cells derived from SAS and H103 cell lines were established using a sphere-forming assay and repeated cisplatin treatments, respectively. The stem cell-like features of the tumour spheres were assessed using flow cytometry, Western blotting and microarray assay. The differences in cisplatin sensitivity of the models were evaluated using cell viability assay after 72 h treatment with cisplatin. Further, mtDNA sequencing was performed using a nanopore sequencer, MinION, to assess the mtDNA mutational and methylation profiles. Meanwhile, the mitochondrial DNA content was quantified using a real-time PCR (qPCR). To correlate the mtDNA alterations with associated functional effects, assessment of mitochondrial respiratory function including evaluation of mitochondrial oxygen consumption rate, mitochondrial membrane potential, and ROS production, were performed. Lastly, HTS was performed via a series of cell viability assays to identify candidate hits that can inhibit the viability of the CSCs model. Potent candidate hits were selected for combinatory testing with cisplatin. The findings showed that the tumour spheres derived from SAS demonstrated stem cell-like features and enhanced cisplatin resistance. qPCR analysis showed that the cells with less mtDNA content were less responsive to cisplatin. A positive selection of m.3910G>C mutation within MT-ND1 gene was significantly observed in the cisplatin-resistant SAS. In comparison to the parental cells, the methylation analysis showed the presence of CpG hypermethylation in the cisplatin-resistant SAS and H103, while SAS tumour spheres showed no methylation changes. Moreover, SAS tumour spheres and the cisplatin-resistant SAS and H103 showed impaired mitochondrial respiratory function and they were less sensitive to ROS-induced cytotoxicity by cisplatin. These findings suggest the relation between mtDNA alterations and the ability of the cells with enhanced cisplatin resistance to profit from mitochondrial dysfunction and evade death, via regulating the extent of ROS production induced by cisplatin. Through HTS, 174 out of 1463 compounds were identified as candidate compounds that inhibited the viability of SAS tumour spheres. Drug combination testing revealed geldanamycin, fimepinostat, and scriptaid as potent compounds that synergized the inhibitory effect of cisplatin against SAS tumour spheres. In short, this preliminary study highlighted the potential role of mtDNA alterations as predictive markers of cisplatin responsiveness in OSCC and the potential use of the compounds in combination therapy with cisplatin against the cisplatin-resistant CSCs for improving the treatment efficacy in OSCC. Further in-depth investigations are crucial for validation purposes.,Ijazah Doktor Falsafah
Pages:	271
Publisher:	UKM, Kuala Lumpur
Appears in Collections:	Faculty of Pharmacy / Fakulti Farmasi

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