Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 70 President St., DD410, Charleston, SC 29425, USA.
Department of Drug Discovery and Experimental Sciences, Medical University of South Carolina, 274 Calhoun Street MSC 141, Charleston, S.C. 29425-1410, USA.
Biochem Pharmacol. 2024 Jan;219:115929. doi: 10.1016/j.bcp.2023.115929. Epub 2023 Nov 22.
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), n-acetyl-L-cysteine, (NAC), or dithiothreitol (DTT). 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 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 and DTT preferentially utilized tricarboxylic acid (TCA) cycle intermediates, while ROT used ketone body substrates such as D, L-β-hydroxybutyric acid. NAC and DTT 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.
还原性应激的特征是细胞电子供体过剩，并与包括癌症在内的各种人类病理相关。我们开发了对还原性应激剂（鱼藤酮（ROT）、N-乙酰-L-半胱氨酸（NAC）或二硫苏糖醇（DTT））具有抗性的黑色素瘤细胞系。抗性细胞分裂得更快，并且细胞内稳态氧化还原偶联比向还原状态转移。抗性引起一般细胞形态的改变，但只有 ROT 细胞的线粒体形态发生了显著变化，数量更多，更孤立、碎片化和肿胀，膜去极化程度更高，网络数量减少。这些变化伴随着基础耗氧量和最大呼吸率的降低。全细胞通量分析和线粒体功能测定表明，NAC 和 DTT 优先利用三羧酸（TCA）循环中间产物，而 ROT 则利用酮体底物，如 D,L-β-羟基丁酸。NAC 和 DTT 细胞的活性氧（ROS）水平持续降低，尽管这伴随着核因子红细胞 2 相关因子 2（Nrf2）的激活，伴随下游基因产物如谷胱甘肽 S-转移酶 P（GSTP）的表达增加。进一步的适应包括内质网蛋白表达的增强，这些蛋白控制未折叠蛋白反应（UPR）。尽管这些 UPR 蛋白在抗性细胞之间的表达模式不同，但趋势表明，每种细胞系对还原性应激的抗性都伴随着持续增加的 UPR 表型。总的来说，肿瘤细胞虽然耐受氧化应激，但可以在致死性还原性应激条件下适应其能量和生存机制。
