Figure 1

DHA reduces viability and induces apoptotic death of human cancer cells. (A) DHA dose-dependently decreases the viability of PA-1, H1299, D54MG and SiHa cells. Cells were treated with the indicated doses of DHA for 24 h, and the cell viability was measured with MTT assays as described in the Materials and Methods. Each bar represents the mean of three determinations repeated in three separate experiments. (B) DHA induces apoptosis. Human cancer cells were incubated with DHA at the indicated doses, and cells were harvested and western blot analysis was performed using PARP, Bax, Bcl-2 and actin antibodies. (C) D54MG cells were incubated for 6, 12, 24 h with indicated doses of DHA, and Sub-G1 DNA contents were evaluated by flow cytometric analysis. Samples were analyzed using FlowJo software. (D) DHA increases the number of TUNEL positive PA-1 cells. Cells were plated on coverslips, and incubated with or without DHA for 6 h. The cells were stained with DeadEnd Fluorometric TUNEL system. Left, the results are shown as a microscopy image. DNA was counterstained with DAPI (scale bar, 200 μm). Right, the percentage of TUNEL positive cells treated with or without DHA was calculated relative to the total number of DAPI-stained nuclei. TUNEL positive cells were counted in three different fields and averaged. ***, P < 0.001. (E) Increases in caspase-3 activities and caspase-3 cleavage formation by DHA. PA-1 cells were treated with various concentrations of DHA for 12 h and lysed. Left, Caspase-3 activity was determined using the fluorogenic substrate DEVD-AFC. Values are mean ± SEM (n = 5). ***, P < 0.001. Right, western blot analysis of cleaved caspase-3, XIAP and Survivin. Equal loading of protein lysate was confirmed using an anti-actin antibody.