中华眼镜蛇L-氨基酸氧化酶分离纯化及其体外抗肿瘤作用-Isolation and Purification of L - Amino Acid Oxidase from Chinese Cobra and Its Antitumor Effect in Vitro.docx

PAGE PAGE 4 inhibitory concentration (IC50) values of NA-LAAO in K562 were 0.80, 0.38 and 0.25 μg/ml at 6 h, 12 h and 24 h, respectively. The IC50 values of NA-LAAO were 1.54, 1.09, 0.48 μg/ml in ECV304 cells, 2.48, 1.74, 0.83 μg/ml in MGC803 cells, 3.18, 1.79, 0.88 μg/ml in U251 cells, 15.4, 6.78, 2.61 μg/ml in A549 cells at 6 h, 12 h and 24 h, respectively. At concentrations lower than 0.625 μg/ml, NA-LAAO did not kill the ECV304 cells but inhibited cell proliferation. Four days exposure to NA-LAAO of 0.156 μg/ml or two days exposure to NA-LAAO of 0.313 μg/ml caused a significant decrease of cell growth. Analysis of ECV304 cell cycle by flow cytometry method indicated the NA-LAAO could increase the G1-phase cells and block the cell cycle in G1-phase. Real-time PCR analysis indicated NA-LAAO up-regulated the mRNA expression of p21, p16, p27 whereas down-regulated the expression of cyclin A2, cyclin B1, cyclin D1, cyclin E1, CDK2, CDK6, Survivin and Skp2. Western-blot analysis indicated NA-LAAO up-regulated p21 and down-regulated cyclin A, CDK2 and CDK6. These effects might contribute to the blockade of cell cycle. Apoptosis-inducing activities and its mechanism of NA-LAAO NA-LAAO inhibited the growth of human bladder carcinoma ECV304 and human leukemia K562 cells and induced apoptosis accompanied DNA hypodiploidy, phosphatidylserine exposure, loss of mitochondrial membrane potential, activation of caspase-9 and caspase-3, and PARP cleavage. NA-LAAO increased the expression of Bax, Hrk, Noxa and Puma after 8 h or 24 h treatment. These results suggested that the mitochondrial pathway is mainly involved in the NA-LAAO-induced apoptosis of ECV304 cells. Pretreatment with z-VAD-fmk rescued cell viability of ECV304 cells at low concentration of NA-LAAO treatment but failed at higher concentration. This result suggested that NA-LAAO might also induce a caspases-independent cell death. The cytotoxicity of NA-LAAO also induced a strong activation of Bcl-2, however, the i