Figure 8. FOXO3-activation enhances MG-2477-induced cell death.

(A) SH-EP/shCtr and SH-EP/shFOXO3-16 cl13 cells were incubated with 20 or 50 nM MG-2477 for 24 hours and cell death was measured by PI-FACS analyses (shown is the mean of three independent experiments). (B) SH-EP/FOXO3 cells were treated with 20 nM MG-2477 alone or in combination with increasing amounts of 4OHT (5 to 20 nM) for 24 hours and analyzed for PI-stained nuclei by flow cytometry. (C) SH-EP/FOXO3-Ctr and SH-EP/FOXO3- Surv cells were pre-incubated with 20 nM 4OHT (24 hours) and/or 100 nM BafA (30 minutes) before addition of 35 nM MG-2477 for another 24 hours. Shown is the mean of four independent experiments. Statistical difference in all experiments was assessed by unpaired t-test (***P<0.001; **P<0.01, *P<0.05). (D) Schematic model of the molecular events triggered by MG-2477 treatment in neuroblastoma cells: In the immediate early phase (red lines), MG-2477 treatment increases NOXA expression, which replaces BECN1 from BCLXL. This triggers ROS downstream of the PI3K-III/BECN1 complex, which results in LC3 conversion, autophagosome formation and autophagic flux. In parallel, Survivin steady state levels decrease, which according to earlier studies from our lab further promotes ROS accumulation. MG-2477 represses the PI3K-I/PKB pathway leading to FOXO3 nuclear accumulation (intermediate phase, blue lines) and to further activation of cellular ROS. In this cell system we have demonstrated before that FOXO3 among other death-related targets induces NOXA and represses Survivin and thereby further perpetuates authophagy and drives the cells into cell death.