Docosahexaenoic Acid Induces Cell Death in Human Non-Small Cell Lung Cancer Cells by Repressing mTOR via AMPK Activation and PI3K/Akt Inhibition

Figure 4

DHA-mediated downregulation of mTOR signaling is related to the induction of autophagy. (a) DHA downregulated mTOR signaling in a dose-dependent manner. A549 (left panel) and H1299 (right panel) cells were incubated with the indicated doses of DHA for 24 h and then subjected to western blot analysis with antibodies against phospho-mTOR, phospho-S6K1, p27, 4E-BP1, and actin. (b) Rapamycin accelerated autophagy and cell death by inhibiting mTOR. A549 cells were incubated for 1 h with or without 1 M rapamycin before incubation for 24 h with 30 M DHA. Cell lysates were prepared and examined by western blotting. (c) DHA reduces PI3K/Akt signaling pathway. Western blotting with antibodies against phosphatidylinositol 3-kinase (PI3K)/Akt signaling molecules showed that DHA downregulates PI3K/Akt signaling in a dose-dependent manner. (d)-(e) Expression of Akt-wt partially rescued DHA-induced NSCLC cell death. pcDNA and a Akt-wt vector were transfected into cells using Lipofectamine 2000 reagent for 12 h. The cells were then exposed to 30 M for another 24 h. Cell viability was examined in an MTT assay ((d) and (e), lower panel) and the cell lysates were analyzed by western blotting with antibodies against phospho-Akt, Akt, phospho-mTOR, and actin ((e), upper panel). . (f) DHA treatment led to a dose-dependent increase in phospho-AMPK levels. A549 (left panel) and H1299 (right panel) cells were treated with indicated doses of DHA for 24 h and cell lysates were examined by western blotting. (g) siAMPK reduced DHA-induced autophagy and inhibited cell death in NSCLC cells by upregulating mTOR signaling. A549 cells were transfected with a siNC or siAMPK and then exposed to 30 M DHA for 24 h. Left panel: western blot analysis of phospho-AMPK, AMPK, phospho-mTOR, LC3-II, and actin expression. Right panel: cell viability was measured in an MTT assay. and .

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