Adaptive changes in tumor cells in response to reductive stress

Reductive stress is characterized by an excess of cellular electron donors and can be linked with various human pathologies including cancer. We developed melanoma cell lines resistant to reductive stress agents: rotenone (ROT^R), n-acetyl-L-cysteine, (NAC^R), or dithiothreitol (DTT^R). Resistant cells divided more rapidly and had intracellular homeostatic redox-couple ratios that were shifted towards the reduced state. Resistance caused alterations in general cell morphology, but only ROT^R cells had significant changes in mitochondrial morphology with higher numbers that were more isolated, fragmented and swollen, with greater membrane depolarization and decreased numbers of networks. These changes were accompanied by lower basal oxygen consumption and maximal respiration rates. Whole cell flux analyses and mitochondrial function assays showed that NAC^R and DTT^R preferentially utilized tricarboxylic acid (TCA) cycle intermediates, while ROT^R used ketone body substrates such as D, L-β-hydroxybutyric acid. NAC^R and DTT^R cells had constitutively decreased levels of reactive oxygen species (ROS), although this was accompanied by activation of nuclear factor erythroid 2-related factor 2 (Nrf2), with concomitant increased expression of the downstream gene products such as glutathione S-transferase P (GSTP). Further adaptations included enhanced expression of endoplasmic reticulum proteins controlling the unfolded protein response (UPR). Although expression patterns of these UPR proteins were distinct between the resistant cells, a trend implied that resistance to reductive stress is accompanied by a constitutively increased UPR phenotype in each line. Overall, tumor cells, although tolerant of oxidative stress, can adapt their energy and survival mechanisms in lethal reductive stress conditions.