K. Lim

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

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).

1021 POSTER Docosahexaenoic Acid Induces Cell Death Through ROS-dependent ERK and JNK Activation in Human Ovarian Cancer Cells

Recent molecular and clinical studies have shown that invasion and metastasis may occur very early in tumour development, thus emphasizing the potential importance of spesific and sensitive detection of circulating tumour cells (CTC). With the ability of refined technologies the identification of CTCs from peripheral blood is emerging as a useful tool for the detection of malignancy, monitoring disease progression and measuring response to therapy. The goal of this study was to identify optimal marker or marker combinations for detection of CTCs in the gastrointestinal malignancies using RT-PCR. Materials and Methods: To detect the presence of CTCs, we analyzed Cytokeratin 19 (CK19), Cytokeratin 20 (CK20) and Mucin 1 (MUC1) mRNA in the peripheral blood of 31 patients with gastrointestinal (gastric, stomach and colorectal) carcinoma and 30 healthy individuals. Results: In RT-PCR analysis of the peripheral blood, 77.4% (24/31), 58.06% (18/31) and 3.22% (1/31) of cancer patients were positive for MUC1, CK20 and CK19 mRNA respectively. The sensivity and specificity for any one of mRNA detected in peripheral blood is 83.3% and 66.6% respectively, with an acurracy of 59%. Conclusions: Our study suggest that MUC1 and CK20 mRNAs in the peripheral blood could be useful molecular markers for gastrointestinal tumours. Combination of these two tumour-spesific mRNA markers would increase the detection rate and may be clinically helpful in predicting the tumour presence and colorectal cancer metastasis.