眼镜蛇毒L-氨基酸氧化酶诱导人胃癌细胞MGC-803细胞凋亡的实验研究

目的: 研究眼镜蛇毒L-氨基酸氧化酶(NA-LAAO)对人胃癌细胞MGC-803的细胞毒性、诱导凋亡作用及可能机制。方法: 采用CCK-8法测定细胞毒性;采用DNA倍体分析和Annexin V/碘化丙啶染色测定细胞凋亡;采用发色底物法测定Caspase-3酶活性;采用Western-blot方法测定聚二磷酸腺苷核糖多聚酸-1(PARP-1)切割。结果: NA-LAAO可抑制MGC-803细胞增殖,6、12、24 h 的IC₅₀分别为(2.48±0.41) 、(1.74±0.27) 和(0.83±0.19) mg/L;NA-LAAO可使细胞DNA分布出现亚二倍体凋亡峰,并可促使细胞膜磷脂酰丝氨酸外翻;NA-LAAO可激活Caspase-3并可促使其底物PARP-1降解。结论: NA-LAAO可能通过激活Caspase-3这一分子机制而诱导细胞凋亡,从而抑制肿瘤细胞增殖。

Evidence of caspases-mediated human stomach carcinoma MGC-803 cells apoptosis induced by L-amino acid oxidase from Naja atra venom

AIM: To investigate the effects and the molecular mechanisms of Naja atra L-amino acid oxidase (NA-LAAO) on the proliferation and cell apoptosis of human stomach cancer MGC-803 cells.METHODS: MGC-803 cells were exposed to different concentrations of NA-LAAO and the cytotoxicity was measured using a CCK-8 kit. The apoptosis-inducing activities of NA-LAAO were examined by propidium iodide staining and Annexin V/PI assay in flow cytometry. The enzyme activity of caspase-3 was measured by a colorimetric assay. The cleavage of PARP-1 was analyzed by Western-blot.RESULTS: NA-LAAO exhibited cytotoxicity on MGC-803 in a dose- and time-dependent manner. The half maximal inhibitory concentration (IC₅₀) values of NA-LAAO were (2.48±0.41), (1.74±0.27) and (0.83±0.19) mg/L in MGC-803 cells at 6 h, 12 h and 24 h, respectively. NA-LAAO induced apoptosis accompanied DNA hypodiploidy, phosphatidylserine exposure, activation of caspase-3, and PARP cleavage.CONCLUSION: NA-LAAO inhibited cell proliferation by inducing a caspase-mediated cell apoptosis.