e., class III phosphatidylinositol-3-kinase). The process of autophagosome formation involves two major steps: nucleation and elongation of the isolation membrane. The Atg1/unc-51-like
kinase (ULK) complex, the autophagy-specific PI3-kinase complex, and PI3P-effectors and their related proteins are important for the nucleation p38 MAPK activity step, whereas the Atg12- and LC3/Atg8-conjugation systems are important for the elongation step. Studies have demonstrated that autophagy plays a wide variety of physiological and pathophysiological roles. Recent evidence has shown that autophagy is associated with cancer pathogenesis and that pharmacologic manipulation of autophagic pathways may represent a new therapeutic strategy for human cancers. However, to date the role of autophagy in cancer is not clearly defined. Although autophagy is a cancer cell survival mechanism against environmental and cellular stresses, it can be associated with cancer cell death under certain situations. Further,
autophagy and apoptosis might be linked to each other and occur simultaneously or sequentially in a cell type-, death stimulus-, and context-dependent manner. Although Hh signaling is known to inhibit cell apoptosis, it remains unknown whether Hh signaling is able to regulate autophagy. The current study describes a novel role of the Hh signaling pathway for regulation of autophagy in human HCC cells. We show that inhibition of the Hh pathway markedly enhanced autophagy http://www.selleckchem.com/products/CP-673451.html through up-regulation of Bnip3 (a member of BH3-only subset of the Bcl-2 family) that displaces Bcl-2 from its binding partner, Beclin-1, and that this process preludes apoptosis. Our findings suggest that the status of autophagy (autophagic flux) is a key factor that may influence cell response to Hh-targeted therapy. Human HCC cells (Huh7, Hep3B, and HepG2) were treated with the Hh signaling ligand, agonists, or inhibitors as indicated and the cells were analyzed for autophagy by immunoblotting
for microtubule-associated Selleckchem Rucaparib protein light chain 3 (LC3) and p62, GFP-LC3 puncta, monodansylcadaverine (MDC) staining, and transmission electron microscopy (TEM). Western blotting analysis was performed to determine the alteration of key signaling molecules including Shh, Patched, Smo, and Gli1, Bnip3, Bcl-2 family proteins, and MEK/ERK1/2. The interaction between Bcl-2 and Beclin-1 was analyzed by immunoprecipitation and immunoblotting assays. For quantitative reverse-transcription polymerase chain reaction (qRT-PCR), total RNA was extracted with the TRIzol plus RNA purification kit and reverse-transcribed to complementary DNA (cDNA) using Superscript II reverse transcriptase; the cDNA samples were used for real-time PCR analysis in triplicate using the QuantiFast SYBR Green PCR kit (Qiagen) on a Bio-Rad C1000 Thermal Cycler.