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

Figure 2

DHA induces autophagy. (a) DHA increased the expression of LC3-II in a dose-dependent manner. A549 (upper panel) and H1299 (lower panel) cells were exposed to the indicated doses of DHA for 24 h. Protein lysates were then prepared, separated in SDS polyacrylamide gels, and immunoblotted with antibodies against LC3-II and actin. (b) Formation of GFP-LC3 puncta in DHA-treated NSCLC cells. A549 cells were transfected with a GFP-LC3 plasmid and then exposed to the indicated doses of DHA for another 24 h before counterstaining with DAPI. Left panel: representative fluorescence microscopy images are shown (scale bar: 2 m). Right: the number of autophagosomes was quantified as the number of GFP-LC3 puncta per transfected cell. Data are expressed as the mean ± SD of ten determinations (each in two separate experiments). . (c) DHA activates autophagic flux in NSCLC cells. Cells were transfected with the GFP-LC3 expression vector using Lipofectamine 2000 reagent for 17 h and then treated with 60 M DHA for another 4 h. DHA-treated cells were then stained with Lysotracker dye. Representative fluorescence microscopy images are shown. Data are expressed as the mean ± SD of five determinations (each in three separate experiments) (scale bar: 10 m). (d) DHA-induced autophagy increases NSCLC cell death. A549 (left) and H1299 (right) cells were incubated for 1 h in the presence or absence of the indicated doses of Bafilomycin and CQ before incubation with indicated doses of DHA for 24 h. Cell lysates were prepared and examined by western blotting.

(a)

(b)

(c)

(d)