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

Abstract

The anticancer properties and mechanism of action of omega-3 polyunsaturated fatty acids (ω3-PUFAs) have been demonstrated in several cancers; however, the mechanism in lung cancer remains unclear. Here, we show that docosahexaenoic acid (DHA), a ω3-PUFA, induced apoptosis and autophagy in non-small cell lung cancer (NSCLC) cells. DHA-induced cell death was accompanied by AMP-activated protein kinase (AMPK) activation and inactivated phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling. Knocking down AMPK and overexpressing Akt increased mTOR activity and attenuated DHA-induced cell death, suggesting that DHA induces cell death via AMPK- and Akt-regulated mTOR inactivation. This was confirmed in Fat-1 transgenic mice, which produce ω3-PUFAs. Lewis lung cancer (LLC) tumor cells implanted into Fat-1 mice showed slower growth, lower phospho-Akt levels, and higher levels of apoptosis and autophagy than cells implanted into wild-type mice. Taken together, these data suggest that DHA-induced apoptosis and autophagy in NSCLC cells are associated with AMPK activation and PI3K/Akt inhibition, which in turn lead to suppression of mTOR; thus ω3-PUFAs may be utilized as potential therapeutic agents for NSCLC treatment.

Figure 1

DHA inhibits cell viability and induces apoptosis in human cancer cells. (a) Upper panel: DHA reduces the viability of A549 and H1299 cells in a dose-dependent manner. Cells were exposed to the indicated doses of DHA for 24 h and the cell viability was measured in an MTT assay. Each bar represents the mean of three determinations. Each experiment was repeated three times. ^∗∗∗ P < 0.001. Lower panel: representative images of A549 and H1299 cells treated with DHA for 24 h (scale bar: 200 μm). (b) DHA induces apoptosis. A549 (upper panel) and H1299 (lower panel) cells were incubated with the indicated doses of DHA for 24 h. The cells were then harvested and western blot analysis was performed with anti-PARP and anti-actin antibodies. (c) Left panel: evaluation of apoptosis by Annexin V staining. Green staining represents Annexin V-positive (apoptotic) cells (scale bar: 50 μm). Right panel: unfixed A549 cells were treated with FITC-Annexin V and then subjected to flow cytometry to examine changes in the plasma membrane. (d) DHA increases the number of TUNEL-positive cells. A549 cells were plated in a 12-well plate containing glass coverslips and then treated with 60 μM DHA for 6 h. Following treatment, apoptosis was detected using the DeadEnd Fluorometric TUNEL System. Left panel: representative fluorescence microscopy images (scale bar: 200 μm). Right panel: the percentage of TUNEL-positive cells in the presence or absence of DHA was expressed relative to the total number of DAPI-stained nuclei. TUNEL-positive cells were counted in three different fields and the numbers averaged. (e) DHA increases the number of NSCLC cells in the sub-G₁ phase. A549 cells were seeded and treated with the indicated doses of DHA for 24 h. The cell-cycle distribution of DHA-treated cells was analyzed by flow cytometry as described in Section 2. Data were analyzed using FlowJo software.

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 μm). 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). ^∗∗∗ P < 0.001. (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 μM 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 μm). (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.

Figure 3

DHA-induced autophagy is required for apoptotic cell death. The indicated cancer cell lines were treated with nontargeting control small interfering RNA (siNC) or siRNAs specific for autophagy-related Atg5 (a) and Atg7 (b). At 18 h after transfection, cells were incubated with the indicated doses of DHA for 24 h. Next, cells were harvested and subjected to western blot analysis with the following antibodies: Atg5, Atg7, LC3-II, and actin (upper panel). Cell viability was measured in an MTT assay (lower panel). ^∗∗ P < 0.01 and ^∗∗∗ P < 0.001. Data are representative of three independent experiments, all with similar results.

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 μM rapamycin before incubation for 24 h with 30 μM 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 μM 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). ^∗∗∗ P < 0.001. (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 μM 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. ^∗ P < 0.05 and ^∗∗∗ P < 0.001.

Figure 5

ω3-PUFAs suppress tumor growth in vivo by inhibiting phospho-Akt and phospho-AMPK, thereby inducing apoptosis and autophagy. (a) Effect of ω3-PUFA on tumorigenicity. Upper panel: LLC cells (3 × 10⁶ cells) were injected subcutaneously into the flanks of wild-type and Fat-1 transgenic mice. Tumor size and volume were monitored every other day for 10 days. Tumor size and volume (middle) were calculated as described in Section 2. Lower panel: hematoxylin and eosin (H&E) staining. ((b)–(e)) Representative fluorescence images showing the TUNEL assay results (b), LC3-II (c), phospho-Akt (Ser473) (d), and phospho-AMPK (e). Fluorescently stained tissues were observed under a fluorescence microscope using DP Controller software (Olympus) for image acquisition. Scale bars: 200 μm.

Figure 6

Schematic model of DHA-induced apoptosis and autophagy in NSCLC cells. DHA-induced autophagy and apoptosis in lung cancer cells are triggered by inhibition of mTOR activation via AMPK activation and PI3K/Akt inhibition.