眼镜蛇毒L-氨基酸氧化酶对人膀胱癌ECV304细胞增殖和细胞周期的影响及机制

摘要

Aim To investigate the effects and the molecular mechanisms of Naja atra L-amino acid oxidase ( NA-LAAO ) on the proliferation and cell cycle of human bladder carcinoma ECV304 cells. Methods ECV304 cells were exposed to different concentrations of NA-LAAO and the cytotoxicity was measured by CCK-8 method. The effects of NA-LAAO on cell cycle were examined by a propidium iodide staining in flow cytometry. The mRNA or protein expressions of cell cycle regulators were determined by real-time PCR or Western blot, respectively. Results NA-LAAO exhibited cytotoxicity in ECV304 in a dose- and time-dependent manner. The half maximal inhibitory concentration ( IC50 ) values of NA-LAAO were ( 1. 54 ± 0. 23 ),( 1. 09 ± 0. 15 ) and ( 0. 48 ± 0. 14 ) mg · L-1 in ECV304 cells at 6 h, 12 h and 24 h, respectively. At concentrations lower than 0. 625 mg · L-1, NA-LAAO did not kill the ECV304 cells but inhibited cellproliferation. Four days ' exposure to NA-LAAO of 0. 156 mg · L-1 or two days' exposure to NA-LAAO of 0. 313 mg · L-1 caused a significant decrease of cell growth. Analysis of ECV304 cell cycle by flow cytometry method indicated that 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, pi 6, p27 whereas down-regulated the expression of cyclin A2, cyclin Bl, cyclin Dl, cyclin El, 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. Conclusion NA-LAAO inhibits cell proliferation by blocking the cell cycle in G1 -phase.%目的 研究眼镜蛇毒L-氨基酸氧化酶(Naja atra L-a-mino acid oxidase,NA-LAAO)对人膀胱癌细胞株ECV304的生长抑制作用,探讨其作用机制.方法 CCK-8法测定NA-LAAO的细胞毒性及对细胞增殖抑制能力;流式细胞术分析NA-LAAO对ECV304细胞周期的影响;实时定量PCR和Western blot分析细胞周期相关基因mRNA和蛋白质表达.结果 NA-LAAO对ECV304细胞有强烈抑制作用,且呈剂量-效应和时间-效应关系.NA-LAAO处理6、12、24 h对ECV304的IC50分别为(1.54±0.23)、(1.09±0.15)和(0.48±0.14)mg·L-1;细胞生长曲线显示,0.156 mg·L-1的NA-LAAO作用4 d可明显抑制ECV304细胞增殖;0.313 mg·L-1从d 2起就有明显效果;0.625 mg·L-1处理后细胞多数死亡;流式细胞术分析提示NA-LAAO可使ECV304细胞阻滞在G1期,并呈量效关系;实时定量PCR结果显示,NA-LAAO可下调Cyclin A2、B1、D1、E1、CDK2、CDK6、Survivin、Skp2,上调p21、p16、p27的表达,Western blot结果显示NA-LAAO可下调Cyclin A、CDK2、CDK6,上调p21,与实时PCR结果一致.结论 NA-LAAO可通过调节细胞周期相关分子,使ECV304细胞阻滞在G1期,抑制细胞增殖.