AMPK-dependent autophagy pathway contributes to deguelin-induced Hep-2 cell death.
2013-01-23T01:07:49Z (GMT) by
<p>Hep-2 cells were pretreated with Z-VAD-fmk (50 µM) or 3-MA (2 mM) for 1 hr before deguelin exposure for 72 hours, cell viability was measured by MTT assay (A). Hep-2 cells were exposed to 100 µM of deguelin for indicated time points, followed by western blot detecting LC3B, tubulin, phospho- and total- level of Ulk1 (B). The association between AMPKα1 and Ulk1 after deguelin exposure (100 µM, 12 h) was determined by IP assay (C–D). Hep-2 cells were transfected with scramble or AMPKα1 siRNA (100 nM) for 48 hours. Western blot was then utilized to test AMPKα expression in transfected cells. Successfully AMPK knocking-down cells and their parental cells were treated with deguelin for indicated time points. AMPK, LC3B, tubulin, phospho- and total- level of Ulk1 were tested by western blots (E), LC3B and phospho-Ulk1 levels were quantified (F). Hep-2 cells were pretreated with 3-MA (2 mM) for 1 hr before deguelin (100 µM) exposure for 72 hours, cell apoptosis was measured by enzyme-linked immunosorbent cell apoptosis assay (G) and hoechst nuclear staining (H). (I) The proposed signaling pathway in this study: deguelin induces both cell apoptosis and autophagy by modulating multiple signaling pathways in cultured HNSCC cells: deguelin inhibits Akt signaling, and down-regulates survivin and cyclin-dependent kinase 4 (Cdk4) expressions, by disrupting their association with Hsp-90. Deguelin induces ceramide production through <i>de novo</i> synthase pathway to promote HNSCC cell death. Ceramide activates AMPK, which directly phosphorylates Ulk1 to promote an early cell autophagy. Cell autophagy is pro-apoptotic in our system. The mean of at least three independent experiments performed in triplicate is shown. Statistical significance was analyzed by ANOVA.</p>