Figure 4

Analysis of generation and effects of hydrogen peroxide by α-ketoglutarate dehydrogenase in vivo and in vitro . (A) In vitro generation of H₂O₂ by KGDH was quantified using Amplex Red oxidation. Co-incubation of KGDH with CPI-613 increased H₂O₂ generation by KGDH in vitro. CPI-157, a lipoate analog lacking in vivo anti-cancer activity, was used as a negative control (see panels B and C). CPI-157 failed to increase in vitro KGDH ROS production. Data are representative of three independent experiments. (**P <0.005 compared to control, ns = not significant compared to control; Student’s t-test; n = 3). (B) CPI-157 stimulates mitochondrial H₂O₂ generation poorly in treated cells as assessed by Prx3 oxidation. (C) CPI-157 is an inactive lipoate analog as assessed by its limited capacity to kill tumor cells. (D) Following siRNA-mediated knockdown of the E3 (dihydrolipoamide dehydrogenase) subunit, H460 cells were exposed to 240 μM CPI-613 for 3 hours and Prx3 oxidation was assayed (left). Assessment of E3 protein levels in siRNA treated cells demonstrates efficient knockdown (right). (E) Quantification of dimer:monomer ratios in panel D using NIH Image-J software **P <0.005 (Student’s t test; n = 3); ***P <0.0005 (Student’s t test; n = 3); ns = not significant. (F) H460 cells treated for 16 hours with 240 μM CPI-613 following siRNA knockdown of E3 were assayed for ATP content using Cell-TiterGlo Kit (Promega). ATP loss under these conditions is diagnostic of cell death[18]. Data are expressed as percent of DMSO control. **P <0.005 (Student’s t test; n = 3). All results representative of at least three experiments. Error bars represent SEM. DMSO, dimethyl sulfoxide; DTT, dithiothreitol.