Passage dependence of NADH redox status and reactive oxygen species level in vitro in triple-negative breast cancer cell lines with different invasiveness

Background: The redox status of nicotinamide adenine dinucleotide (NAD; including oxidized form NAD⁺ and reduced form NADH) plays key roles in both health and disease and has been actively studied to develop cancer biomarkers and therapeutic strategies. With the optical redox imaging (ORI) technique, we have been investigating the relationship of NADH redox status, reactive oxygen species (ROS), and invasiveness in breast cancer cell cultures, and have associated higher invasiveness with more oxidized NADH redox state. However, the cell cultures may have phenotypic drift and metabolic change with increased passage numbers.

Methods: We investigated the passage-dependence of NADH redox status and ROS levels in two triple-negative breast cancer (TNBC) cell cultures: the more invasive/metastatic MDA-MB-231 and the less invasive/metastatic HCC1806 cell lines. We measured the NADH redox status, redox plasticity, and cytoplasmic and mitochondrial ROS levels under the basal condition and metabolic perturbations of the mitochondrial electron transport chain. We evaluated the dependence of redox and ROS profiles on the cell passage number by comparing the early (<20 passages) with the late (>60 passages) passage cells.

Results: (I) NADH redox and ROS baselines are stable and independent of cell passage number, but can vary with passage number under metabolic perturbations depending on specific perturbation and cell line; (II) NADH redox status and intracellular ROS levels can change discordantly in cancer cells; (III) under both basal and metabolically perturbed conditions, the more invasive cell line has a more oxidized NADH redox status with a higher basal cytoplasmic ROS level than the less invasive line, regardless of passage number.

Conclusions: The general correlation between redox, ROS, and invasiveness in studied TNBC cells is not very sensitive to passage number. These results indicate that NADH redox and basal ROS status in TNBC likely reflect the intrinsic progressive nature of TNBC cells.