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

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.


Introduction
Lung cancer is the main cause of cancer-related death worldwide. According to the latest statistics from the United States National Cancer Institute, it is estimated that 224,210 Americans will be diagnosed with lung cancer in 2014 [1]. There are two types of lung cancer, namely, small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC), with more than 80% of lung cancer cases being NSCLC [2]. Because NSCLC is much less sensitive to chemotherapy than SCLC [3], a new approach for treating NSCLC is required.
Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid ( 3-PUFA), induces apoptosis in tumor cells by regulating several key signaling mediators, including Wnt/ -catenin [19], p53-regulated activator protein 1 [20], and mitogen-activated protein kinase [21,22]. In addition, we have previously reported that DHA simultaneously induces apoptosis and autophagy in human cervical cancer as well as prostate cancer cells, and this process involves mTOR repression [23,24]. Despite several studies describing that the anti-lung cancer activity of DHA may be dependent on its proapoptosis effect [21,[25][26][27], it is still unclear whether it also induces autophagy in vitro and in vivo.
Here, we examined the mechanism(s) underlying DHAinduced cell death in human NSCLC cells. The results showed that DHA reduced cell viability and induced both apoptosis and autophagy. Moreover, DHA-induced cell death was associated with AKT-mTOR signaling inhibition and AMPK activation. Similarly, lung cancer cells implanted into Fat-1 transgenic mice exhibited higher levels of apoptosis, a higher autophagy index, and lower levels of phospho-AKT than cells implanted into wild-type mice. Taken together, these data show that DHA induces apoptosis and autophagy through AKT-mTOR signaling inhibition and AMPK activation, suggesting that 3-PUFAs may be a potential treatment for human NSCLC.

Cells Lines and Cultures.
Human NSCLC A549 cells and H1299 cells were purchased from American Type Cell Culture Collection (Rockville, MD, USA) and maintained in Dulbecco's Modified Eagle's Medium (DMEM; GIBCO, Grand Island, NY, USA) supplemented with 10% heatinactivated fetal bovine serum (FBS; GIBCO), penicillin, and streptomycin (GIBCO) in a humidified 5% CO 2 atmosphere at 37 ∘ C.

Cell Viability Assay.
Cell viability was determined using thiazolyl blue tetrazolium bromide (MTT; Sigma). Cells were seeded into 96-well plates (7 × 10 3 per well) and incubated for 18 h at 37 ∘ C to allow adherence. The cells were then incubated with serum-free medium for 24 h and then treated with DHA for another 24 h. The cells were then incubated with MTT for 2 h and the formazan products dissolved in DMSO. Absorbance was assayed at 570 nm in a spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Western Blot Analysis.
Western blot analysis was performed as described previously [28]. Briefly, cell lysates (30 g) were resolved by 6-15% sodium dodecyl sulfatepolyacrylamide gels and then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). The membranes were then blocked in 5% (w/v) skimmed-milk for 1 h, followed by incubation with appropriate primary antibodies (diluted 1 : 1000-1 : 5000) overnight at 4 ∘ C. Bound antibodies were detected with peroxidase-conjugated goat anti-rabbit or goat anti-mouse secondary antibodies and the blots developed using enhanced chemiluminescence (Millipore).

Apoptosis Analysis.
Apoptosis was measured using a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, flow cytometry, and an Annexin V assay as described previously [23,29].
2.6. Immunofluorescence Analysis. Cells were grown to 70% confluence in growth medium for 18 h before infection with recombinant adenoviruses expressing GFP-tagged LC3 (GFP-LC3, a gift from Professor Chang Deok Kim, Chungnam National University, Korea). DHA was then added to the infected cell cultures for 24 h. The cells were observed by an Olympus iX70 fluorescence microscope.

Small Interfering RNAs (siRNAs) and
Transfection. siR-NAs targeting Atg5 and Atg7 were purchased from Invitrogen (Camarillo, CA, USA). A nontargeting control siRNA was purchased from Bioneer (Daejeon, Korea). Cells For the transfection of expression vectors, cells were grown to 70-80% confluence and then switched to serumfree medium for 2-4 h before being transiently transfected with HA-tagged myr-Akt1 (Akt-wt) and HA-tagged kinasedead (K179M) dominant negative Akt1 (Akt-dd) (kindly provided by Dr. Incheol Shin; Hanyang University, Korea) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions. After 12-18 h, the cells were subjected to different treatments. Control transfections were carried out using an empty pcDNA3 vector.

Tumor Xenograft Study.
Transgenic Fat-1 C57BL/6 mice were provided by Dr. J. X. Kang (Harvard University, USA). Control C57BL/6 mice were purchased from Central Lab Animal Inc. (Seoul, Korea). The control and Fat-1 C57BL/6 mice were kept under specific pathogen-free conditions and received care according to the guidelines of the Institutional Animal Care and Use Committee of Chungnam National University. The Fat-1 C57BL/6 mice ( = 5) used in this study were heterozygous, male, and 6 weeks old at the time of the experiments. Each mouse was subcutaneously injected with Lewis lung cancer (LLC) cells (3 × 10 6 in 100 L serum-free DMEM) on day 0. The tumor size was measured every other day using calipers for 10 days. Tumor size was calculated as length × wide and volume was calculated as 0.5 × length × (wide) 2 .

Statistical Analysis.
Statistical analyses were done as recommended by an independent analyst. These included the unpaired Student's -test. All values are expressed as mean ± SD, and statistical significance was accepted for values of <0.05. * , * * , and * * * mean < 0.05, < 0.01, and < 0.001, respectively.

DHA Reduces Cell Viability and Induces Apoptosis in
Human NSCLC Cells. It has been shown previously that DHA induces apoptotic cell death in several types of cancer cells [27,[30][31][32]. To examine whether DHA induces apoptosis in human NSCLC cells, we tested the effect of DHA on the viability of A549 and H1299 cells in vitro. DHA reduced the viability of both cell lines (Figure 1(a), upper). Moreover, when observed under a light microscope, DHA-treated cells appeared shrunken, rounded, and detached from the culture dishes ( Figure 1(a), lower), characteristics suggestive of apoptosis.
To investigate whether this observed DHA-induced reduction in cell viability was actually due to apoptosis, we first examined the cleavage of PARP, an apoptotic marker.
DHA treatment led to an increase in the levels of cleaved PARP in both A549 and H1299 cells (Figure 1(b)). Furthermore, DHA treatment caused a marked increase in the number of Annexin V-positive cells (Figure 1(c)), which is another early apoptotic indicator [33]. We next performed a TUNEL assay and cell-cycle analysis to look for nuclear DNAstrand breaks and hypodiploid DNA formation because both are increased during apoptosis [34]. DHA treatment increased the number of TUNEL-positive A549 cells (Figure 1(d)) and the percentage of A549 cells in the sub-G1 phase (Figure 1(e)). Taken together, these data indicate that DHA induces apoptosis in NSCLC cells.

DHA Triggers Autophagy as a Prerequisite for Apoptotic
Cell Death. Apoptosis and autophagy are highly interconnected [35], and our previous studies showed that DHA activates both of these cascades simultaneously in cervical and prostate cancer cells [23,24]. To determine whether autophagy is also involved in DHA-induced NSCLC cell death, we initially measured the expression LC3-II (the membrane-bound lipidated form of LC3), a biomarker of autophagic initiation [36]. Western blot analysis revealed that DHA caused a remarkable increase in LC3-II expression (Figure 2(a)). Similarly, DHA led to a dose-dependent increase in the number of GFP-LC3 puncta per virally infected A549 cell (Figure 2(b)). It is known that during autophagy, autophagosomes fuse with lysosomes in which both LC3-II and the cargo are degraded [23]. To examine whether DHA interferes with autophagic flux, we therefore next examined the colocalization of lipidated LC3-II with lysosomes using Lysotracker dye. DHA treatment led to a marked increase in the colocalization of lipidated LC3-II with Lysotracker compared with that in control cells (Figure 2(c)), suggesting that DHA does not block autophagosome-lysosome fusion. To further confirm this, A549 cells were exposed to DHA in the absence or presence of CQ and Bafilomycin A1 (inhibitors of lysosomal acidification) and LC3-II levels were analyzed by immunoblotting (Figure 2(d)). We found that the DHAinduced increase in LC3-II expression was further increased by CQ and Bafilomycin A1. Taken together, these results indicate that autophagy is activated and may play a role in DHA-induced cell death.
To unveil the relationship between DHA-autophagy and DHA-apoptotic cell death, we used siRNAs to knock down two essential autophagy gene products, Atg5 and Atg7. Although silencing of Atg5 and Atg7 had no effect on the viability of A549 cells, it strongly suppressed DHA-induced autophagy and apoptosis as evidenced by a reduction in LC3-II and viability (Figures 3(a) and 3(b)). These results imply that autophagy, at least partially, contributes to DHA-induced apoptotic cell death.
Autophagy has been shown to be essential for cell survival under certain stressful conditions. For example, hypoxia and the anticancer drug, Nelfinavir, induce autophagy by inactivating the growth factor receptors and by suppressing Akt signaling [37,38], both of which play a positive role in cell survival; however, our data indicate that DHA induces autophagy, which enhances cell death. This observation is  in line with other reports showing that Clioquinol and Rhabdastrellic acid-A promote cell death in hepatocellular carcinoma, lung adenocarcinoma, myeloma, and leukemia cells lines by inducing autophagy [36,39]. Although the exact mechanism by which autophagy contributes to DHAinduced apoptosis in NSCLC cells is still unclear, it is known that autophagy can promote cell death by selectively eliminating vital components, such as mitochondria and peroxisomes [40]. We have reported that DHA-induced apoptosis is associated with mitochondrial damage [24,31]; therefore, it is possible that DHA-induced autophagy is triggered by eliminating components that are essential for survival, such as mitochondria.

DHA-Mediated Downregulation of mTOR Signaling Is
Associated with Autophagy Induction. mTOR is the key negative regulator of autophagy [4]. To examine whether mTOR inhibition is involved in DHA-induced autophagy, we investigated the expression of mTOR signaling-related molecules in NSCLC cells after DHA treatment. DHA reduced the levels of phospho-mTOR in both A549 (Figure 4(a), left panel) and H1299 (Figure 4(a), right panel) cells, indicating that the activity of mTOR is repressed by DHA. Consistent with this, the levels of mTOR's two readout molecules, phospho-S6K1 and phospho-4E-BP1, were also found to be decreased in A549 cells. Meanwhile, increases in p27 (whose activation is indicative of mTOR inhibition) [23] were observed in DHAtreated A549 and H1299 cells (Figure 4(a)), suggesting that DHA indeed suppresses the mTOR signaling pathway. Next, to confirm the role of mTOR in DHA-induced cell death, we pretreated A549 cells with rapamycin followed by DHA. We found that DHA-induced decreases in uncleaved PARP and increases in LC3-II expression in A549 cells were enhanced by pretreatment with rapamycin (Figure 4(b)). These results imply that DHA-induced autophagy and apoptosis are associated with mTOR inhibition. mTOR is directly linked to PI3K/Akt signaling [41], and the PI3K/Akt/mTOR signaling pathway plays an important role in cell proliferation and survival [42]. We then asked whether PI3K/Akt is associated with DHA-induced mTOR  inactivation. To test this, we examined the expression of PI3K/Akt signaling molecules and found that DHA induced a marked reduction in PI3K, Akt, and phospho-PTEN (a negative regulator of the PI3K) (Figure 4(c)). Next, to obtain evidence for the interconnection between decreased PI3K/Akt signaling and DHA-induced cell death, we overexpressed Akt in A549 cells before DHA treatment (overexpression in itself had no significant effect on the cell viability of A549 cells) (Figure 4(d)). DHA treatment led to a reduction in cell viability and increased the levels of phospho-mTOR and LC3-II; however, these phenomena were partially reversed by Akt overexpression (Figure 4(e)). These data suggest that DHA-induced cell death is also associated with Akt inhibition in NSCLC cells. Evidence suggests that DHA can disrupt the association between lipid rafts and epidermal growth factor receptor (EGFR), leading to inactivation of EGFR and its downstream PI3K/Akt signaling in lung cancer cells [43]. Accordingly, it is reasonable to speculate that DNA might inhibit the PI3K/Akt signaling pathway by disrupting EGFR phosphorylation and its association with lipid rafts; however, further studies are needed to investigate whether this is the mechanism underlying DHA-mediated Akt inactivation. In addition to Akt, another key mediator of mTOR is AMPK, which has been demonstrated to negatively regulate mTOR activity [15]. We sought subsequently to determine whether DHA-induced mTOR inactivation involves AMPK in NSCLC cells. To this end, we first measured the levels of AMPK in DHA-treatment A549 and H1299 cells. DHA treatment led to increased AMPK activity in both A549 and H1299 cells, as evidenced by the promoted expression levels of phospho-AMPK (Figure 4(f)). Importantly, knockdown of AMPK not only rescued DHA-induced mTOR inactivation, but also inhibited DHA-induced autophagy and cell death in A549 cells (Figure 4(g)). These results demonstrated that DHA-induced autophagy mediated by mTOR inhibition, at least partly, attributes to AMPK activation. Previously, endoplasmic reticulum (ER) stress has been shown to elevate AMPK levels, leading to the upregulation of Ca 2+ -CaMKK signaling and autophagy-related genes such as Atg5 and Atg12 [44]. We found that the levels of phosphoeukaryotic translation initiation factor-2 , which reflects ER stress,  increased in DHA-treated A549 cells in a time-dependent manner (data not shown). Thus, although the exact mechanism underlying DHA-mediated AMPK activation remains unknown, it is possible that AMPK activation in DHA-treated cells is induced by the activation of Ca 2+ -CaMKK and autophagy-related genes via ER stress.

3-PUFAs Suppress Tumor Growth In Vivo by Inhibiting the Expression of Phospho-Akt and Phospho-AMPK, Thereby
Inducing Apoptosis and Autophagy. The in vitro results presented above show that DHA reduced the viability of human NSCLC cells. We next examined the effect of DHA on tumor formation and growth in Fat-1 transgenic mice. Fat-1 transgenic mice express 3-desaturase and thus produce higher levels of 3-PUFAs than wild-type (WT) mice [45]. The mouse origined NSCLC, LLC cells were subcutaneously injected into WT mice and Fat-1 transgenic mice, and tumor size and volume were measured. We found that both the size and volume of the tumors in Fat-1 transgenic mice were markedly lower than those in WT mice ( Figure 5(a)), suggesting that 3-PUFAs suppress the growth of NSCLC cells in vivo. To verify whether apoptosis and autophagy played a role in this process, we performed TUNEL assays and indirect immunofluorescence assays on tumor tissue sections to examine apoptosis and autophagy levels, respectively. The number of TUNEL-positive cells and the number of LC3-II puncta were higher in Fat-1 tumors than in WT tumors ( Figures 5(b) and 5(c)), indicating that both apoptosis and autophagy played a role in inhibiting tumor growth in Fat-1 transgenic mice. Next, to determine whether PI3K/Akt and AMPK signaling were responsible for the increased levels of apoptosis and autophagy, we examined the levels of Akt and AMPK in tumor tissues by immunohistochemistry. As shown in Figures 5(d) and 5(e), the levels of phospho-Akt (Ser473) decreased and the levels of phospho-AMPK increased in Fat-1 tumor tissues. Together, these data demonstrate that 3-PUFAs induce apoptosis and autophagy in vivo by regulating AMPK and PI3K/Akt signaling.

Conclusions
In conclusion, we describe for the first time that DHA triggers autophagy and apoptosis in NSCLC cells, which simultaneously promotes cell death. Our results indicate that the DHA-induced autophagy and apoptosis are controlled by repressing mTOR through AMPK activation and PI3K/Akt inhibition ( Figure 6). These data suggest that DHA may represent a potentially useful reagent for treating human NSCLC in clinical settings.