Selective killing of cancer cells harboring mutant RAS by concomitant inhibition of NADPH oxidase and glutathione biosynthesis

摘要

A–C qRT-PCR analysis of the expression levels of GCLC, GCLM, and GSS genes in T72 and T72Ras cells. β-actin serves as a control. D Western blot analysis of p22phox, GCLC, and GR protein levels in T72 and T72Ras cells. β-actin serves as a loading control. E–F Quantitative analysis of GCLC and GR protein levels in D. G T72 parental cells and their isogenic HRASG12V-transformed T72Ras cancer cells were treated with glutathione biosynthesis inhibitor BSO at 0, 10, 30, or 100 μM for 30 h. Cell viability was measured by flow cytometry with annexin-V/PI double staining. The numbers shown at the upper left of each panel indicate the percentage of the annexin-V/PI positive cells. H T72 and T72Ras cells were treated with DPI at 1 or 3 μM for 30 h. Cell viability was measured as described in G, and cell death was expressed as the percentage of the annexin-V/PI positive cells. I Combined treatment using BSO (10, 30, or 100 μM) and DPI (1 μM) for 30 h induced significant death of T72Ras cells (lower panel) but not parental T72 cells (upper panel). Data are mean ± SD of three independent experiments with Student’s t test. **p < 0.01, ***p < 0.001, ****p < 0.0001. KDa kilodalton.