Investigation of mechanisms involved in mitochondrial dysfunction mediated by dihydroorotate dehydrogenase inhibitors in breast cancer cell lines / Muhammad Aiman Akmal Shahhiran

Muhammad Aiman Akmal , Shahhiran (2024) Investigation of mechanisms involved in mitochondrial dysfunction mediated by dihydroorotate dehydrogenase inhibitors in breast cancer cell lines / Muhammad Aiman Akmal Shahhiran. Masters thesis, Universiti Malaya.

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Abstract

Breast cancer has recorded an incidence rate surpassing lung cancer in the recent global report, making it as the most prominent cancer worldwide. DHODH inhibitors are studied for cancer therapy because of their capacity in delivering cell cycle arrest as well as other various therapeutic potential in affecting ATP production, membrane potential and ROS in cancer cells. In this study, the mechanisms of action of brequinar sodium and teriflunomide were explored with a specific focus on their effects on the mitochondrial bioenergetics in breast cancer cell lines with various receptor status. Cell proliferation inhibition and cell cycle analysis were analysed using MTT assay and PI staining, respectively. Analysis of intracellular and mitochondrial ROS were conducted using ROS detection assay kits. The biochemical assays of respiratory complexes activities were performed spectrophotometrically using mitochondrial complexes assay kits I-IV. Inhibition assay showed that MCF-7 and MDAMB-231 cells of the subtypes (ER+/PR+/HER2-, HR+) and (ER-/PR-/HER2-, TNBC), respectively, were responsive to brequinar but not SKBR-3 (ER-/PR-/HER2+, HER2+). Meanwhile, all cancer cells of different subtypes were less responsive to teriflunomide without showing a competitive value of IC50. The normal cell line was also responsive to both DHODH inhibitors, showing that they are not cancer specific. In cell cycle analysis, all responsive cells showed cell cycle arrest at G1/S phase with a higher percentage of cells in S-phase compared to their respective DMSO controls. In Western blot analysis, p130 cellular differentiation biomarker was found to increase in poorly differentiated MDAMB-231 treated with DHODH inhibitors. DHODH expression was also highest in this TNBC cell line that showed the highest sensitivity towards DHODH inhibition. For ROS analysis, both intracellular and mitochondrial ROS were increased in all treated breast cancer cells compared to normal, regardless of their sensitivities to DHODH inhibitors. The complex I activity was affected by DHODH inhibitor in all breast cancer cells without any particular link to the receptor status. DHODH inhibition disrupted the activities of complex II in HER2+ and complex III in HR+ and TNBC cells. The complex IV activity was not affected by the inhibitors but was rather disrupted as a consequence of dysfunctional complex III. In conclusion, the sensitivity of breast cancer cells towards DHODH inhibitors is independent of their ER/PR receptor status but has a closer association with HER2 receptor status. DHODH inhibitors cause cell differentiation in poorly differentiated breast cells. The sensitivity of breast cancer cells to DHODH inhibitors was found to associate with DHODH expression, doubling time, receptor and tumorigenic status. Most interestingly, we have found that MDAMB-231 TNBC cell was the most affected by DHODH inhibition in terms of sensitivity, cell cycle arrest, induction of cell differentiation, production of ROS and mitochondrial complexes disruption. This study has provided a strong motive to integrate DHODH inhibitors in a combinatorial approach as a potential and valuable targeted therapy for the TNBC subtype.

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