As shown in Fig. 4A, we observed robust up-regulation of the BH3-only protein, Bnip3, by GANT61 in all three HCC cell lines; the expression of other Bcl-2 family proteins (including Bim, Noxa, Puma, Bcl2, and Bclxl) were not significantly affected, although the level of Mcl-1 was SB203580 slightly reduced in two of the three HCC cell lines. GANT61 induced a 4.39-fold, 2.84-fold, and 1.97-fold increase in Bnip3 mRNA level in Huh7, Hep3B, and HepG2 cells, respectively, as determined by qRT-PCR (Fig. 4B). The effect of GANT61 on Bnip3 expression was dose-dependent (at 24- and 48-hour timepoints) (Fig. 4C). The role of Bnip3 in GANT61-induced autophagy was supported by the observations that siRNA knockdown

of Bnip3 prevented GANT61-induced LC3II accumulation (Fig. 4D, left panel) and that overexpression of Bnip3 enhanced GANT61-induced LC3II accumulation (Fig. 4D, right panel) and reversed SAG-induced LC3II reduction (Fig. 4E). We sought to further investigate the mechanism by which Hh signaling regulates Bnip3. As the Bnip3 promoter does not contain the Gli consensus DNA-binding sequences, it is likely that Hh signaling might regulate Bnip3 through an indirect mechanism. Given that Bnip3 is a downstream target of the MEK/ERK signaling pathway[11, 12]

and that Hh and MEK/ERK signaling pathways are known to interconnect in other cells,[13-15] we performed experiments Epacadostat order to determine whether inhibition of Hh by GANT61 might induce Bnip3 expression by way of activation of MEK/ERK. As shown in Fig. 4F, GANT61 treatment increased the phosphorylation of MEK and ERK1/2

(but did not Protein kinase N1 affect the levels of total MEK and ERK1/2). We observed that inhibition of MEK by U0126 prevented GANT61-induced phosphorylation of ERK1/2, expression of Bnip3, and accumulation of LC3II (Fig. 4G). These findings suggest that GANT61-induced Bnip3 expression is mediated at least in part through activation of the MEK/ERK pathway. Although Bnip expression is known to be regulated by nuclear factor kappa B (NF-κB),[16] p53,[17] and DNA methyltransferase-1 (DNMT-1),[18] these molecules were not altered by GANT61 treatment in our system (Supporting Fig. S3). Beclin-1, the mammalian ortholog of yeast Atg6, is a well-known key regulator of autophagy; it is a critical component of the class III phosphatidylinositol-3-kinase complex (PI3KC3) required for autophagy. The overall structure of Beclin-1/Atg6 and its essential role in autophagosome formation is evolutionarily conserved throughout all eukaryotic phyla. Whereas Beclin-1 expression promotes autophagy, Beclin-1 reduction decreases autophagic activity.[19] Sequence alignment and structural modeling indicate that Beclin-1 contains a putative BH3-like domain (amino acids 112-123), which is known as a novel BH-3 domain only protein.[20] The BH-3 domain of Beclin-1 interacts with Bcl-2, which leads to inhibition of autophagy.