ABSTRACT
BACKGROUND: Associations between mitochondrial genetic abnormalities (variations and copy number, i.e. mtDNAcn, change) and elevated ROS have been reported in cancer compared to normal cells. Since excessive levels of ROS can trigger apoptosis, treating cancer cells with ROS-stimulating agents may enhance their death. This study aimed to investigate the link between baseline ROS levels and mitochondrial genetic abnormalities, and how mtDNA abnormalities might be used to predict cancer cells' response to ROS-stimulating therapy. METHODS: Intracellular and mitochondrial specific-ROS levels were measured using the DCFDA and MitoSOX probes, respectively, in four cancer and one non-cancerous cell lines. Cells were treated with ROS-stimulating agents (cisplatin and dequalinium) and the IC50s were determined using the MTS assay. Sanger sequencing and qPCR were conducted to screen the complete mitochondrial genome for variations and to relatively quantify mtDNAcn, respectively. Non-synonymous variations were subjected to 3-dimensional (3D) protein structural mapping and analysis. RESULTS: Our data revealed novel significant associations between the total number of variations in the mitochondrial respiratory chain (MRC) complex I and III genes, mtDNAcn, ROS levels, and ROS-associated drug response. Furthermore, functional variations in complexes I/III correlated significantly and positively with mtDNAcn, ROS levels and drug resistance, indicating they might mechanistically influence these parameters in cancer cells. CONCLUSIONS: Our findings suggest that mtDNAcn and complexes I/III functional variations have the potential to be efficient biomarkers to predict ROS-stimulating therapy efficacy in the future.
Subject(s)
Antineoplastic Agents/pharmacology , DNA, Mitochondrial , Mitochondria/drug effects , Mitochondria/genetics , Mitochondria/metabolism , Oxidation-Reduction/drug effects , Reactive Oxygen Species/metabolism , Antineoplastic Agents/chemistry , Binding Sites , DNA Copy Number Variations , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Electron Transport Complex I/chemistry , Electron Transport Complex I/metabolism , Electron Transport Complex III/chemistry , Electron Transport Complex III/metabolism , High-Throughput Nucleotide Sequencing , Humans , Models, Molecular , Molecular Conformation , Protein Binding , Structure-Activity RelationshipABSTRACT
BACKGROUND: Mitochondria are considered a primary intracellular site of reactive oxygen species (ROS) generation. Generally, cancer cells with mitochondrial genetic abnormalities (copy number change and mutations) have escalated ROS levels compared to normal cells. Since high levels of ROS can trigger apoptosis, treating cancer cells with low doses of mitochondria-targeting / ROS-stimulating agents may offer cancer-specific therapy. This study aimed to investigate how baseline ROS levels might influence cancer cells' response to ROS-stimulating therapy. METHODS: Four cancer and one normal cell lines were treated with a conventional drug (cisplatin) and a mitochondria-targeting agent (dequalinium chloride hydrate) separately and jointly. Cell viability was assessed and drug combination synergisms were indicated by the combination index (CI). Mitochondrial DNA copy number (mtDNAcn), ROS and mitochondrial membrane potential (MMP) were measured, and the relative expression levels of the genes and proteins involved in ROS-mediated apoptosis pathways were also investigated. RESULTS: Our data showed a correlation between the baseline ROS level, mtDNAcn and drug sensitivity in the tested cells. Synergistic effect of both drugs was also observed with ROS being the key contributor in cell death. CONCLUSIONS: Our findings suggest that mitochondria-targeting therapy could be more effective compared to conventional treatments. In addition, cancer cells with low levels of ROS may be more sensitive to the treatment, while cells with high levels of ROS may be more resistant. Doubtlessly, further studies employing a wider range of cell lines and in vivo experiments are needed to validate our results. However, this study provides an insight into understanding the influence of intracellular ROS on drug sensitivity, and may lead to the development of new therapeutic strategies to improve efficacy of anticancer therapy.
