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AMPK-dependent autophagy pathway contributes to deguelin-induced Hep-2 cell death.

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posted on 2013-01-23, 01:07 authored by Yan-li Yang, Chao Ji, Zhi-gang Bi, Chun-cheng Lu, Rong Wang, Bing Gu, Lei Cheng

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 de novo 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.

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