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


getmorefigures.php?uid=PMC4538321&req=5

fig4: 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.

Mentions: 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 DHA-treated 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.


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

Show All Figures
getmorefigures.php?uid=PMC4538321&req=5

fig4: 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.
Mentions: 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 DHA-treated 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.

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