Mitochondria-targeted OXPHOS inhibition enhances radiotherapy efficacy by disrupting mitochondrial function

Reducing tumor cell oxygen consumption has emerged as a promising strategy to counter hypoxia-induced radioresistance in solid tumors. Previously, we found that OXPHOS inhibition using PEGylated mitochondria-targeted atovaquone (Mito-PEG-ATO) and mitochondria-targeted tamoxifen (MitoTam) alleviated hypoxia in tumor spheroids. Here, we investigated the underlying metabolic and redox-related mechanisms-of-action and examined whether mitochondria-targeted OXPHOS inhibition enhances radiotherapy (RT)-induced DNA damage.

The metabolic and redox-related effects of Mito-PEG-ATO and MitoTam alone or combined with RT were examined in B16OVA, MOC1.3D5 and MC38 cells or clones containing an HRE-eGFP-ODD construct. Viability was assessed using a CCK-8 assay on 2D cells. ROS levels were measured after treatment with Mito-PEG-ATO and MitoTam for 24 h with CellRox green and fluorescence monitoring via IncuCyte Zoom. Antioxidant capacity was measured after OXPHOS inhibition relative to Trolox, an analogue of Vitamin E used as antioxidant standard, and mitochondrial membrane potential (MMP) was examined using MitoTracker Orange. Intracellular ATP and metabolic dehydrogenase activity was assessed in tumor spheroids via CellTiter-Glo and CCK-8 assays. Viability, MMP, ATP levels and metabolic activity were measured at 4 h and 24 h post-RT. DNA damage was quantified by γH2AX immunofluorescence in spheroids. Tumor hypoxia following treatment with Mito-PEG-ATO was determined with immunohistochemistry in MOC1.3D5 tumor-bearing mice.

Mito-PEG-ATO and MitoTam reduced cell viability independent of hypoxia and increased ROS production, which was most pronounced for Mito-PEG-ATO, while no effect was observed on antioxidant capacity. Mitochondrial function was impaired by OXPHOS inhibition, shown by MMP disruption, ATP depletion and reduced metabolic activity, with no further impairment upon combination with RT. However, combining Mito-PEG-ATO or MitoTam with RT increased DNA damage compared to either treatment alone. Treatment with Mito-PEG-ATO in MOC1.3D5 tumor-bearing mice did not alleviate hypoxia or alter lactate levels.

Mito-PEG-ATO and MitoTam increase ROS production and disrupt mitochondrial function, enhancing RT-induced DNA damage and potentially improving RT efficacy. However, mice treated with Mito-PEG-ATO did not show a reduction in tumor hypoxia.