138 Inhibition of MTor Through AMPK Activation and PI3K/Akt Inhibition is Important to Docosahexaenoic Acid-induced Cell Death in Human Non-small Cell Lung Cancer Cells

Abstract

Introduction: Reactive oxygen species (ROS) produced by docosahexaenoic acid (DHA) have an important function in cancer cell death. However, the exact mechanism of ROS production, after DHA stimulation, is not clearly understood. Here, we determined that elevated levels of ROS generated by mitochondrial respiration is directly associated with DHA-induced cervical cancer cell death. Material and Method: The effects of DHA on cell proliferation and cell cycle were examined by MTT assay and FACS. DHA-induced apoptosis was analyzed using the TUNEL assay, caspase activity assay, and western blot. Dihydroethidium (DHE) was used for reactive oxygen species (ROS) measurement in cytosol. MitoSox was performed for ROS measurement in mitochondria. Results and Discussion: The levels of caspase 3 activity, TUNELpositive staining cells and Sub-G1 portion were markedly increased in DHA-treated cancer cells, suggesting that apoptosis is responsible for the DHA-induced cervical cancer cell death. Furthermore, DHA was able to induce both mitochondrial complex I substrateand complex II substratesupported mitochondrial ROS production in isolated mitochondria from rodent liver. Meanwhile, a reduction in oxygen consumption rate and an increase in mitochondrial ROS production as measured by MitoSOX, were also observed in DHA-treated cancer cells, indicating that DHA can directly act on mitochondrial respiration and enhance ROS generation. The role of DHA-induced mitochondrial ROS production in apoptosis was further identified by the findings that DHA reduced the mitochondrial membrane potential, resulting in cardiolipin oxidation and cytochrome c release from mitochondria, and that N-acetylcysteine, an antioxidant almost completely blocked these processes as well as ROS production occurred in mitochondria and remarkably reversed the apoptotic cell death triggered by DHA. Conclusion: From the results presented here, we conclude that mitochondria actively participate in the DHA-induced apoptotic cell death by the generation of mitochondrial ROS. This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Education, Science and Technology (2011– 0006232 and 2011–0003060).