AbstractMitochondria are considered a primary intracellular site of reactive oxygen species (ROS) generation. Mitochondrial genetic abnormalities (mutations and copy number change) have been reported in various cancer types that might be associated with elevated ROS levels in cancer cells compared to normal cells. Since high levels of ROS can trigger apoptosis, treating cancer cells with ROS-stimulating agents may enhance cell death. This study aimed to investigate: 1) how baseline ROS levels might influence cancer cells’ response to ROS-stimulating therapy; 2) the link between mitochondrial genetic abnormalities and baseline ROS levels; 3) how specific mitochondrial DNA mutations might be utilised to predict cancer cells’ response to ROS-stimulating therapy.
Sanger sequencing was used to screen for mitochondrial DNA (mtDNA) mutations and qPCR to measure mtDNA copy number (mtDNAcn) in four cancer and one noncancerous cell lines. Three-dimensional structural mapping and analysis of all the nonsynonymous mutations were then performed to assess their functional importance. The overall baseline intracellular ROS levels and mitochondrial superoxide were measured using the 2’,7’-dichlorofluorescin diacetate assay and MitoSOX indicator, respectively. Cells were treated with a conventional drug (cisplatin) and a mitochondria-targeting agent (dequalinium chloride hydrate) separately and jointly. Cell viability was assessed using MTS assays and drug combination synergisms were analysed using the Combination Index method. Apoptosis events were detected and the relative expression levels of the genes and proteins involved in ROS-mediated apoptosis pathways were also investigated.
Greater numbers of non-synonymous mutations in the mitochondrial complexes I/III coding regions were detected in cancer cells with higher ROS levels. Our data showed a positive correlation between the baseline ROS levels, number of non-synonymous mutations, mtDNAcn and drug resistance levels in the tested cells. Synergistic effect of both drugs was also observed with ROS being the key contributor in cell death.
Our findings assert that cancer cells with low levels of ROS are more sensitive to ROSstimulating agents, while cells with high levels of ROS are more resistant. Moreover, specific mutations in the complexes I/III coding regions, A10398G, T11120C, C12084T, A13681G, G13708A, C13802T, A13966G and T14798C, could be used to indicate elevated intracellular ROS and therefore to predict drug resistance. Patient-derived primary cell cultures and tissues, and in vivo experiments will be required to validate the present findings.
|Date of Award||Apr 2020|
|Supervisor||Qian An (Supervisor), James Smith (Supervisor) & Karen Ball (Supervisor)|