目的 探讨天然产物槐定碱对人肺癌A549细胞系体外增殖和侵袭的抑制作用及其机制。方法 应用噻唑盐(MTS)法检测槐定碱对肿瘤细胞生长的作用,流式细胞术检测槐定碱对肿瘤细胞凋亡、细胞周期和细胞内活性氧(ROS)生成的变化,Transwell试验检测肿瘤细胞体外侵袭能力的变化,黏附实验检测肿瘤细胞体外黏附能力的变化,Realtime PCR法和Western blotting法检测细胞凋亡相关基因Survivin、Bcl-2和caspase-3/8、细胞周期相关基因CDK-2、黏附分子CD44和基质金属蛋白酶MMP-2/9的表达以及AKT磷酸化的变化;报告基因技术检测NF-κB转录活性和细胞内泛素化蛋白酶体的活性的变化。结果 槐定碱对肺癌A549细胞具有明显的抑制作用,经过槐定碱作用后,肿瘤细胞内活性氧水平和凋亡率上调,细胞周期阻滞在G2/M期,细胞体外侵袭和黏附能力降低,Realtime PCR和Western blotting结果显示,促凋亡蛋白caspase-3/8表达上调,抑制凋亡蛋白Survivin和Bcl-2,细胞周期相关蛋白CDK-2、黏附分子CD44以及基质金属蛋白酶MMP-2/9表达下调,AKT磷酸化水平下调,NF-κB转录活性和细胞内泛素化蛋白酶体的活性降低。结论 槐定碱可抑制肺癌A549细胞体外增殖和侵袭能力,可能与槐定碱提升肿瘤细胞内的活性氧水平、促进其凋亡、阻滞细胞周期、抑制泛素化蛋白酶体的活性和调节肿瘤相关基因表达有关。
Abstract
OBJECTIVE To investigate the effect and mechanism of allomatrine in proliferation and invasion in vitro inhibition of human lung cancer A549 cell line. METHODS After treatment with allomatrine, MTS assay was employed to determine the proliferation of cancer cells, flow cytometry to determine the apoptosis rate, cell cycle distribution and intracellular ROS production, transwell assay to determine the cell invasion potential in vitro, adhesion assay to determine the cell adhesion potential in vitro, realtime PCR and Western blotting assay to determine the expression of apoptosis related gene Survivin, Bcl-2 and caspase-3/8, the cell cycle related protein CDK-2, adhesion molecule CD44, matrix metalloproteinases MMP-2/9 and the phosphorylation of AKT, report gene assay to determine the transcription activity of NF-κB and the activity of ubiquitin proteasome. RESULTS Allomatrine significantly inhibited the proliferation and invasion in vitro of A549 cells, flow cytometry showed that apoptosis rate and ROS production was increased and the cell cycle was halted by G2/M phase, the expression of pro-apoptosis gene caspase-3/8 was upregulated while the anti-apoptosis proteins Survivin and Bcl-2 was downregulated, the expression of CDK-2, CD44 and MMP-2/9 was downregulated, and the phosphorylation of AKT was downregulated, the transcription activity of NF-κB and the activity of ubiquitin proteasome were inhibited after Allomatrine treatment. CONCLUSIONS Allomatrine was able to inhibit proliferation and invasion in vitro of human lung cancer A549 cell line by inducing ROS production, promoting apoptosis, arresting cell cycle, inhibiting ubiquitin proteasome and regulating tumor related gene expression.
关键词
槐定碱 /
肺癌 /
侵袭 /
增殖
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Key words
allomatrine /
lung cancer /
invasion /
proliferation
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中图分类号：
R965
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参考文献
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脚注
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基金
国家自然基金青年项目资助(81303267)
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