苯甲酰乌头原碱对人肺癌A549细胞自噬和凋亡的影响
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篇名: 苯甲酰乌头原碱对人肺癌A549细胞自噬和凋亡的影响
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摘要: 目的:研究苯甲酰乌头原碱(BAC)对人肺癌A549细胞自噬和凋亡的影响,探讨其用于非小细胞肺癌治疗的作用机制。方法:采用不同剂量的BAC(10、50、100、200、400 μmol/L)分别对A549细胞进行处理后,观察细胞的形态变化,并采用CCK-8法测定细胞的增殖抑制率。将细胞分为对照组(不加药物)和BAC低、高剂量组(200、400 μmol/L),分别加入相应药物处理后,采用流式细胞术测定细胞凋亡率,采用聚合酶链式反应法和Western blotting测定细胞中凋亡相关因子B淋巴细胞瘤因子2(Bcl-2)、Bcl-2凋亡相关X蛋白(Bax)、胱天蛋白酶3(Caspase-3)和自噬相关因子Beclin1、LC3、P62的基因及蛋白表达水平。结果:采用不同剂量的BAC处理后,细胞出现皱缩、排列稀疏、溶解等现象,BAC 100、200、400 μmol/L剂量组细胞增殖抑制率显著升高(P<0.05或P<0.01)。流式细胞术检测结果显示,BAC低、高剂量组细胞凋亡率分别在药物作用24、48 h时有不同程度的上升,且该促凋亡作用具有剂量、时间依赖趋势。与对照组比较,BAC各剂量组细胞中Bcl-2、P62的mRNA和蛋白表达水平均有不同程度的降低,Bax、Caspase-3、Beclin1、LC3的mRNA和Bax、Active Caspase-3、P62、Beclin1、LC3Ⅱ/Ⅰ的蛋白表达水平均有不同程度的升高,其中BAC低剂量组Caspase-3的mRNA和Bcl-2、Active Caspase-3、Beclin1、LC3Ⅱ/Ⅰ、P62蛋白表达水平,以及BAC高剂量组各目标基因的mRNA和蛋白的表达水平差异均有统计学意义(P<0.05或P<0.01),且上述影响均呈剂量依赖趋势。结论:BAC可抑制A549细胞的增殖、促进其凋亡,并且能促进Beclin1、LC3(LC3Ⅱ/Ⅰ)、Bax、Caspase-3(Active Caspase-3)表达和抑制P62、Bcl-2等自噬/凋亡相关基因及蛋白的表达;其机制可能与BAC通过促进细胞发生过度自噬从而导致细胞凋亡有关。
ABSTRACT: OBJECTIVE: To study the effects of benzoyl aconitine (BAC) on autophagy and apoptosis of human lung cancer A549 cells, and to investigate its mechanism in anti-non-small cell lung cancer. METHODS: A549 cells were treated with different doses of BAC (10, 50, 100, 200, 400 μmol/L), and then cell morphology was obtained; the proliferation inhibition rate of the cell was determined by CCK-8 assay. The cells were divided into control group (without drug), BAC low-dose and high-dose groups (200, 400 μmol/L). After treated with relevant drugs, the apoptosis rate of cells was determined by flow cytometry. The gene and protein expression of apoptosis-related factors Bcl-2, Bax, Caspase-3 as well as autophagy-related factors Beclin1, LC3, P62 were determined by RT-PCR and Western blotting assay. RESULTS: After treated with different doses of BAC, the cells were shrunken and sparsely arranged; inhibitory rate of cell proliferation was increased significantly in BAC 100, 200, 400 μmol/L groups (P<0.05 or P<0.01). Results of flow cytometry showed that the apoptotic rates of cells were increased to different extents in BAC low-dose and high-dose groups after treated for 24 and 48 h, in a concentration and time-dependent manner. Compared with control group, mRNA and protein expression of Bcl-2 and P62 were decreased to different extents in BAC groups; mRNA expression of Bax, Caspase-3, Beclin1 and LC3 as well as protein expression of Bax, Active caspase-3, P62, Beclin1, LC3Ⅱ/Ⅰ were increased to different extent; there was statistical significance in mRNA expression of Caspase-3, and protein expression of Bcl-2, Active Caspase-3, Beclin1, LC3Ⅱ/Ⅰ and P62 in BAC low-dose group as well as all target mRNA and protein expression in BAC high-dose group (P<0.05 or P<0.01), in dose-dependent manner. CONCLUSIONS: BAC can inhibit the proliferation and promote the apoptosis of A549 cells, promote Beclin1, LC3(LC3Ⅱ/Ⅰ),Bax and Caspase-3 (Active Caspase-3) gene and their protein expression, but inhibit P62 and Bcl-2 gene and their protein expression. The mechanism may be related to BAC inducing apoptosis by promoting excessive autophagy of cells.
期刊: 2019年第30卷第20期
作者: 邵鑫,韩彬,蒋先虹,黎风,何梅,刘福
AUTHORS: SHAO Xin,HAN Bin,JIANG Xianhong,LI Feng,HE Mei,LIU Fu
关键字: 苯甲酰乌头原碱;非小细胞肺癌;A549细胞;增殖;自噬;凋亡
KEYWORDS: Benzoyl aconitine; Non-small cell lung cancer; A549 cells; Proliferation; Autophagy; Apoptosis
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