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Studies on Antitumor Activities of Metabolites Produced by Pseudomonas Aeruginosa

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Tutor: YangShuLin
School: Nanjing University of Technology and Engineering
Course: Biochemical Engineering
Keywords: Pseudomonas aeruginosa,di-rhamnolipid,pyocyanin,antitumor activity,oxidative str
CLC: R96
Type: Master's thesis
Year:  2014
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Pseudomonas aeruginosa is a common gram-negative bacterium. This bacterium can produce secondary metabolites which show good application future in environment, medicine and industry. As a kind of biosurfactant with extensive use, rhamnolipid still show obvious biological activities, such as speeding wound healing, inhibiting alga growth, antibacterial activity, and so on. Pyocyanin is a blue phenazine pigment with strong redox activity. Pyocyanin can easily penetrate membranes, produce excess free radical and result in cell death. In this dissertation, the high rhamnolipid producing strain Pseudomonas aeruginosa Ml4808mutated by simulated space condition was used for fermentation. Secondary metabolites were purified, and the chemical structure of rhamnolipid was characterized. Then, the antitumor activities and mechanism of rhamnolipids and pyocyanin were investigated. The main results are as follows:1. The main constituent of rhamnolipids purified from fermentation supernatant was di-rhamnolipid (Rh2C10C10). The substance reduced viability of MCF-7cells significantly and increased the activity of senescence-associated β-galactosidase. The results of AO/EB staining, DNA ladder, caspase-3activity and the expressions of p53, bax, bcl-2, casepase-3, caspase-9, Fas genes indicated that, di-rhamnolipid induced cell necrosis.2. Pyocyanin was purified from seed solution. Pyocyanin treatment resulted in a concentration-dependent decrease in A549and HepG2cells. AO/EB staining, DNA ladder and single cell gel electrophoresis indicated that pyocyanin induced apoptosis. Further research showed that pyocyanin evoked a dose-related elevation of intracellular ROS and caspase-3activity, and contents of major antioxidants were changed. Simultaneously, the mRNA expressions of p53, caspase-9, bax and PIG3in HepG2increased, and the ratio of bcl-2/bax decreased. N-acetyl-L-cysteine (NAC) pretreatment markedly enhanced viability of A549and HepG2cells and repaired the level of oxidative stress markers and genes expressions. On the whole, pyocyanin induced oxidative stress in A549and HepG2cells, and resulted in p53and mitochondria-mediated apoptosis in HepG2cells.
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