Figure 5

CPI-613 inhibits α-ketoglutarate dehydrogenase activity selectively in tumor cells. (A, B) Flux through KGDH was assayed in H460 (A) and Bx-PC3 (B) tumor cells using pulse delivery of 1-¹⁴C-labeled glutamate, whose labeled carbon is released as CO₂ by KGDH. Each flux panel is coupled on the right with a parallel experiment demonstrating that commitment to and execution of cell death (measured by ATP levels, before or after 3 hours of recovery from drug treatment) occurs well after times used for flux analysis. (C) Steady-state metabolite analysis of BxPC-3 tumor cells after 2-hour treatment with 240 μM CPI-613 (shaded boxes) compared to mock-treated samples (open boxes) shows decreases inTCA cycle intermediates citrate, succinate, fumarate and malate and increases in anaplerotic inputs alanine, aspartate and glutamine. (D) Diagram of the TCA cycle, its two main carbon entry points, and anaplerotic transaminations supporting alanine and aspartate catabolism. (E, F) Normal HBT cells show no increase in Prx3 oxidation (E) and only a slight, transient inhibition of KGDH flux (F) under conditions producing robust effects in lung cancer cells. Vehicle control in panel F refers to the tumor cells; the HBT vehicle control behaved similarly. All results are representative of at least three experiments or (panel C) data point collections. Error bars represent SEM except for panel C in which they represent 95% confidence limits. Box plots (panel C) are used to convey the spread of the data with the middle 50 % of the data represented by boxes and whiskers reporting the range of the data. The solid bar across the box represents the median value of those measured while the + is the mean. Any outliers are shown as dots outside the whiskers of the plot. DTT, dithiothreitol; HBT cells, primary human bronchial/tracheal epithelial cells; KGDH, α-ketoglutarate dehydrogenase.