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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 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.
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fig1: 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.

Mentions: It has been shown previously that DHA induces apoptotic cell death in several types of cancer cells [27, 30–32]. 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.


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

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

fig1: 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.
Mentions: It has been shown previously that DHA induces apoptotic cell death in several types of cancer cells [27, 30–32]. 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.

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