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

Kim N, Jeong S, Jing K, Shin S, Kim S, Heo JY, Kweon GR, Park SK, Wu T, Park JI, Lim K - Biomed Res Int (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea ; Infection Signaling Network Research Center, School of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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fig2: 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.

Mentions: 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 DHA-induced 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.


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

Kim N, Jeong S, Jing K, Shin S, Kim S, Heo JY, Kweon GR, Park SK, Wu T, Park JI, Lim K - Biomed Res Int (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: 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.
Mentions: 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 DHA-induced 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.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, School of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea ; Infection Signaling Network Research Center, School of Medicine, Chungnam National University, Daejeon 301-747, Republic of Korea.

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.

No MeSH data available.


Related in: MedlinePlus