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

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. . Lower panel: representative images of A549 and H1299 cells treated with DHA for 24 h (scale bar: 200 m). (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 m). 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 M DHA for 6 h. Following treatment, apoptosis was detected using the DeadEnd Fluorometric TUNEL System. Left panel: representative fluorescence microscopy images (scale bar: 200 m). 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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