Fig. 5: The small molecule inhibitor ATN-224 inhibits SOD1 activity, sensitizes GB cells for hypoxia-induced cell death and the combination with rapamycin abrogates the protective effect of mTORC1 monotherapy.

A LN-229 and T98G cells were treated with the SOD1 inhibitor ATN-224 in serumfree DMEM and analyzed by SOD1/2 enzyme activity assay. ATN-224 treatment decreased SOD1 activity up to 99%. B LN-229 and T98G cells were preincubated with ATN-224 for 24 h in serumfree DMEM containing 25 mM glucose. Afterwards cells were treated with the SOD1 inhibitor ATN-224 in serumfree DMEM containing 2 mM glucose in normoxia or hypoxia (0.1%) for 25 h (LN-229) and 16 h (T98G). Cells were analyzed by H2DCFDA and PI staining. At this timepoint no relevant cell death was observed (data not shown) and only PI negative ( = living) cells were quantified for H2DCFDA FACS analysis (n = 4, mean ± SEM, two-way ANOVA followed by Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001). C LN-229 and T98G cells were treated with the SOD1 inhibitor ATN-224 (Veh. (DMSO), 0.1 µM, 0.5 µM, 1 µM, 5 µM, 10 µM, 20 µM) in serumfree DMEM in normoxia for 96 h. Cell density was analyzed by CV staining. D LN-229 and T98G cells were seeded (500 cells per well) in 6-well-plates and after 24 h treated with the SOD1 inhibitor ATN-224 with a concentration of 0.01 µM, 0.1 µM and 1 µM. Cell growth of the clones was stopped via CV staining after 10 days (n = 3, one-way ANOVA followed by Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001). E LN-229 and T98G cells were preincubated with ATN-224 for 24 h in serumfree DMEM containing 25 mM glucose. Afterwards cells were treated with the SOD1 inhibitor ATN-224 in serumfree DMEM containing 2 mM glucose in normoxia or hypoxia (0.1%) for 22 h (LN-229) and 10 h (T98G). Cells were analyzed by PI staining (n = 4, mean ± SEM, two-way ANOVA followed by Tukey’s multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001).