The role of autophagy and nitric oxide in arsenite-induced cytotoxicity and genotoxicity in lung adenocarcinoma A549 cells

• Main Authors: Ahmed Magdy Abu El-Fotouh, 馬格帝
• Other Authors: Yang, Jia-Ling
• Format: Others
• Language: en_US
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/72655293049335266488

碩士 === 國立清華大學 === 生物科技研究所 === 104 === It is well known that arsenic induces cytotoxicity and genotoxicity in mammalian cells, and arsenic-induced autophagy was clearly demonstrated. Autophagy is an evolutionarily conserved, catabolic process that maintains cellular homeostasis by regulating organelles and proteins turnover and, meantime, induces autophagic cell death. In line with this, nitric oxide is also known for its dual role in cancer cells by regulating cell survival and cell death. We have recently identified that arsenite can induce nitric oxide progression in A549 lung adenocarcinoma cell line. So, I focused my thesis studies on understanding the role of autophagy and nitric oxide mediated by arsenite in cytotoxicity and genotoxicity in A549 non-small cell lung cancer cells. To accomplish the previously stated goal, I have performed the acridine orange staining assay to detect the activation of autophagosome formation, and used western blot technique to monitor the conversion of LC3B-I to LC3B-II, which is a key marker protein for autophagy induction. The preliminary results from fluorescent microscopy have been further validated by western blot analysis, which confirmed the role of arsenite in inducing autophagy. Moreover, the results obtained from MTT assay and colony forming ability assay showed that treating the cells with autophagy inhibitors, such as spautin-1, chloroquine, or 3-methyladenine, enhanced arsenite-induced cell death suggesting that autophagy has a protective role under arsenite stress. In addition, to study the role of nitric oxide in regulating cell viability under arsenite treatment; I treated the cells with nitric oxide modulators, such as PTIO, 1400W, and GSNO, and the results obtained from MTT assay suggested that NO has a prosurvival role against arsenite toxicity. Moreover, investigating the potential function of autophagy and nitric oxide in genome stability via assessing the ability of the cells to form micronuclei was also conducted, and the results suggested that arsenite-induced autophagy is required for suppressing genotoxicity following arsenite exposure. Interestingly, the results obtained from micronucleus assay suggested that nitric oxide is critically required for genome stability in normal and stressful conditions.