Fig. 6. Inhibition of autophagy potentiates the antiproliferative effect of GANT61.

SW1736 cells seeded in 96-well plates (2000 cells/well) were incubated with the indicated concentrations of GANT61 minus or plus CQ (10 μM) (A) for 72 h or 5Z (2 or 5 μM) (B) for 48 h. Cell viability was measured by using a CellTiter-Glo kit. Data are the mean ± SD of the triplicate from one representative of three independent experiments with similar results. **p < 0.01, compared to the untreated control; ^##p < 0.01, compared to the counterparts in SW1736 left untreated or treated with the same concentrations of GANT61. C The mechanisms of Shh pathway inhibition-induced autophagy. Shh binding to Ptch releases the restriction of Smo activity. Activated Smo disrupts the interaction between Sufu and Gli1. Inhibition of the Shh pathway by the Smo inhibitor cyclopamine or by the Gli1 inhibitor GANT61 activates TAK1, which then activates JNK and AMPK. JNK promotes autophagy by phosphorylating Bcl-2 and dissociating it from Beclin-1. Free Beclin-1 becomes available for the initiation of autophagy. AMPK phosphorylates ULK1 and activates it. ULK1 is involved in the assembly of the pre-initiation complex and the early formation of autophagosomes. Activation of the autophagic pathway by inhibition of the Shh pathway attenuates apoptosis by multiple pathways including the activation of NF-kB and JNK by TAK1. Autophagy itself appears to also suppress apoptosis, albeit its underlying mechanism remains unknown. In addition, it is also not clear how Gli1 suppression leads to TAK1 activation.