Abstract 3005: The impact of mitochondrially targeted oncology agents on mitochondrial DNA (mtDNA) integrity

Abstract

Abstract Reactive oxygen species (ROS) are natural byproducts of mitochondrial oxidative phosphorylation. Dysfunction in electron shuttling leads to enhanced ROS production that is notorious for inflicting damage to macromolecules. Mitochondrial DNA (mtDNA) is particularly sensitive to ROS as it is located in close proximity to the respiratory chain. Additionally, mtDNA lack histones which are known to provide protection from ROS. Mitochondria also have limited DNA repair mechanisms making damage to mtDNA more detrimental to mitochondrial physiology. Recent evidence suggests that oxidative insult, leads to mtDNA degradation to prevent the accumulation of mutagenic base lesions, which is enabled by the high redundancy of mtDNA. The primary objectives of this study were to determine the impact of redox-active mitochondrial-targeted chemotherapeutic agents on mitochondrial physiology and mtDNA integrity. All experiments were performed in two cancer cell lines [MDA-MB-231 (breast) and H23 (lung)] that were treated with 2 uM of the mitochondrial-targeted redox-active compounds: MitoTempol [C4 (MT4)], MitoQuinone (MQ) and MitoChromanol-Acetate (MCA) for 1 or 24 hrs. Mitochondrial status was determined by measuring mitochondrial membrane potential, superoxide production, oxygen consumption rate (OCR) and protein levels of the respiratory chain complexes using the JC-1 and MitoSOX assays, Seahorse XF analysis, and immunoblotting, respectively. Next, mtDNA integrity was analyzed by quantifying the degree of mtDNA fragmentation using PCR amplification of a 10kb fragment compared to a short 100bp fragment. Moreover, changes in the mtDNA replication machinery were assessed using immunoblotting. MQ and MCA induced a loss in mitochondrial function and mtDNA replication machinery as indicated by a reduction in membrane potential, OCR and protein levels. Furthermore, increases in superoxide levels and mtDNA fragmentation were observed with MQ and MCA treatment. Finally, we assessed mitochondrial content using two methods. The first method being the ratio of mitochondrial to nuclear DNA, determined by qPCR of short (∼100bp) nuclear and mitochondrial sequences. The second method was the ratio of mitochondrial outer membrane proteins to cytoplasmic housekeeping proteins, determined by immunoblotting. Both mitochondria DNA and outer membrane proteins decreased with MQ and MCA treatment suggesting that redox-active mitochondrial-targeted agents may reduce the mitochondrial content in cancer cells. Citation Format: Kaytee L. Pokrzywinski, Thomas G. Biel, Dmitry Kryndushkin, V. Ashutosh Rao. The impact of mitochondrially targeted oncology agents on mitochondrial DNA (mtDNA) integrity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3005.