Figure 4.

l-Cystine import induces oxidative stress and ROS-mediated cell death in glucose-deprived cancer cells. A, l-cystine, but not l-cysteine, import results in glucose deprivation–induced cell death. Glucose deprivation–sensitive T98 cells were cultured in l-cystine–free medium and subjected to glucose deprivation for 8 h. l-Cystine starvation rescued T98 cells from glucose deprivation–induced cell death. Addition of l-cystine (200 μm), but not l-cysteine (200 μm), sensitized T98 cells to glucose deprivation. B, γ-GCS contributes resistance to glucose and l-cystine deprivation. Glucose deprivation–sensitive T98 cells were starved of glucose and l-cystine for 24 h. Addition of 200 μm γ-GCS conferred a modest resistance compared with DMSO control. ** denotes p value <0.01 by Student's t test (n = 3). C, l-cystine induces oxidative stress upon glucose deprivation. T98 cells were deprived of glucose in l-cystine–free medium for 3 h, and metabolites were quantified using LC-MS metabolomics. Addition of l-cystine (200 μm), but not l-cysteine (200 μm), induced GSH depletion and oxidative stress as measured by the ratio of GSH/GSSG and NADPH/NADP⁺. D, ROS accumulation following glucose deprivation is driven by l-cystine import. T98-sensitive cells were deprived of glucose in the presence or absence of l-cystine for 3 h, and ROS levels were measured by flow cytometry by CM-H₂DCFDA staining. Cells that had been starved of glucose and l-cystine for 3 h were then re-supplemented with l-cystine (200 μm), l-cysteine (200 μm), or neither for an additional 3 h. Left, mean fluorescent intensity of CM-H₂DCFDA signal. Center, histograms of CM-H₂DCFDA for T98 cells cultured with glucose, without glucose but with l-cystine, or without glucose or l-cystine for 3 h. Right, histograms of CM-H₂DCFDA intensity for T98 cells starved of glucose and l-cystine for 3 h and then re-supplemented with l-cystine, l-cysteine, or neither for an additional 3 h. E, l-cystine import induces redox imbalance upon glucose deprivation. Glucose deprivation–sensitive T98 cells were starved of glucose for 3 h in the presence and absence of l-cystine, at which point l-cystine (200 μm) was added for 10, 30, and 60 min. Addition of l-cystine induced a redox imbalance as indicated by dramatic decreases in the ratios GSH/GSSG and NADPH/NADP⁺. Error bars are standard deviation of the mean.