Mitochondrial DNA abnormalities provide mechanistic insight and predict reactive oxygen species-stimulating drug efficacy
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
Supplementary information. Figure S1. Representative image of agarose gel electrophoresis of PCR products. Figure S2. Correlation between the D-loop variations and the baseline intracellular ROS level, mitochondrial superoxide level, drug resistance against CDDP and DQA, relative content of mtDNA and the total number of variations. Figure S3. Correlation between the total variations and the baseline intracellular ROS level, mitochondrial superoxide level, drug resistance against CDDP and DQA and relative content of mtDNA. Table S1. List of common variations identified in the 5 cell lines. Table S2. List of unique variations identified in the 5 cell lines. Table S3. Summary of frequency in healthy tissues and heritability of functional variations identified in the 5 cell lines. Table S4. Summary of correlations between mtDNA parameters and ROS-stimulating drug IC50s, intracellular and mitochondrial ROS. Table S5. List of the primers designed to amplify 17 overlapping mtDNA fragments.
Posted 16 Dec, 2020
On 21 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
Invitations sent on 15 Dec, 2020
On 15 Dec, 2020
On 15 Dec, 2020
On 15 Dec, 2020
On 23 Nov, 2020
Mitochondrial DNA abnormalities provide mechanistic insight and predict reactive oxygen species-stimulating drug efficacy
Posted 16 Dec, 2020
On 21 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
Received 06 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 05 Jan, 2021
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
On 24 Dec, 2020
Invitations sent on 15 Dec, 2020
On 15 Dec, 2020
On 15 Dec, 2020
On 15 Dec, 2020
On 23 Nov, 2020
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8