亚硒酸钠通过Keap1/Nrf2/ARE信号通路诱导人肺腺癌A549细胞凋亡
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摘要
目的探究亚硒酸钠诱导人肺腺癌A549细胞凋亡的作用及可能的分子机制。方法 MTT法检测不同浓度亚硒酸钠对人肺腺癌A549细胞的抑制增殖率;倒置荧光显微镜观察细胞经亚硒酸钠处理后的形态学改变;流式细胞术检测细胞凋亡率;激光共聚焦观察活性氧荧光强度;多功能酶标仪测定氧化应激参数含量;Western blot检测Keap1/Nrf2/ARE信号通路蛋白的表达,以及Nrf2在细胞质和细胞核中的蛋白表达情况。结果亚硒酸钠剂量依赖性地抑制人肺腺癌A549细胞增殖,亚硒酸钠作用于人肺腺癌A549细胞24 h后,通过Hoechst 33342固定和流式细胞术分析,细胞凋亡率明显增加;亚硒酸钠可使活性氧和丙二醛水平明显升高,还原型谷胱甘肽和超氧化物歧化酶含量明显下降;同时亚硒酸钠能够下调Keap1、Nrf2和HO-1蛋白的表达,并且抑制Nrf2发生核位移。结论亚硒酸钠通过抑制人肺腺癌A549细胞增殖,诱导细胞凋亡,调节细胞内的氧化应激反应,调控Keap1/Nrf2/ARE抗氧化信号通路,从而促进肿瘤细胞凋亡。
Aim To study the induction of apoptotic effect of sodium selenite on human lung cancer A549 cells and its mechanisms.Methods A549 cells were exposed to different concentrations of sodium selenite for 24 h. MTT assay was applied to determine A549 cell proliferation. Inverted fluorescence microscope was used to investigate the morphological changes in A549 cells. Flow cytometry analysis was applied to assess the apoptotic rates of A549 cells. Laser confocal microscope was employed to measure the reactive oxygen species(ROS) fluorescence intensity. A multi-detection reader was used to determine the antioxidant parameter. Western blot was utilized to detect the expression of Keap1, Nrf2, HO-1 and Nrf2 in cytoplasm and nucleus.Results MTT results showed that sodium selenite inhibited the proliferation of A549 cells in a concentration-dependent manner. After treatment with sodium selenite for 24 h, the apoptotic rate of A549 cells was markedly increased through Hoechst 33342 staining and flow cytometry measurement. Sodium selenite significantly up-regulated ROS and malondialdehyde(MDA) content and down-regulated the levels of superoxide dismutase(SOD) and glutathione(GSH). Meanwhile, sodium selenite treatment also reduced the expressions of Keap1, Nrf2 and HO-1 at protein levels and inhibited Nrf2 protein nuclear translocation in A549 cells.Conclusions Treatment with sodium selenite induces A549 cells apoptosis, which may contribute to the anti-proliferation activity, induction of apoptosis and regulation of oxidative stress reaction and Keap1/Nrf2/ARE antioxidative signaling pathway expression.
Aim To study the induction of apoptotic effect of sodium selenite on human lung cancer A549 cells and its mechanisms.Methods A549 cells were exposed to different concentrations of sodium selenite for 24 h. MTT assay was applied to determine A549 cell proliferation. Inverted fluorescence microscope was used to investigate the morphological changes in A549 cells. Flow cytometry analysis was applied to assess the apoptotic rates of A549 cells. Laser confocal microscope was employed to measure the reactive oxygen species(ROS) fluorescence intensity. A multi-detection reader was used to determine the antioxidant parameter. Western blot was utilized to detect the expression of Keap1, Nrf2, HO-1 and Nrf2 in cytoplasm and nucleus.Results MTT results showed that sodium selenite inhibited the proliferation of A549 cells in a concentration-dependent manner. After treatment with sodium selenite for 24 h, the apoptotic rate of A549 cells was markedly increased through Hoechst 33342 staining and flow cytometry measurement. Sodium selenite significantly up-regulated ROS and malondialdehyde(MDA) content and down-regulated the levels of superoxide dismutase(SOD) and glutathione(GSH). Meanwhile, sodium selenite treatment also reduced the expressions of Keap1, Nrf2 and HO-1 at protein levels and inhibited Nrf2 protein nuclear translocation in A549 cells.Conclusions Treatment with sodium selenite induces A549 cells apoptosis, which may contribute to the anti-proliferation activity, induction of apoptosis and regulation of oxidative stress reaction and Keap1/Nrf2/ARE antioxidative signaling pathway expression.
引文
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[14] 孟哲,腾帅,王琛,等. 积雪草酸通过Nrf2抗氧化通路抑制自发性高血压大鼠心肌纤维化[J]. 中国药理学通报,2018,34(8):1073-8. Meng Z, Teng S, Wang C, et al. Asiatic acid inhibits cardiac fibrosis via activating Nrf2-mediated antioxidant signaling pathway in spontaneous hypertensive rats[J]. Chin Pharmacol Bull, 2018, 34(8): 1073-8.
[2] Yang T, Cao C, Yang J, et al. miR-200a-5p regulates myocardial necroptosis induced by Se deficiency via targeting RNF11[J].Redox Biol, 2018, 15: 159-69.
[3] 田崇梅,夏道宗,邢梦雨. 土茯苓总黄酮与亚硒酸钠联用对铅离子螯合能力及体外抗氧化性的研究[J]. 中华中医药学刊,2018,36(6):1392-5. Tian C M, Xia D Z, Xing M Y. Study on combination of total flavonoids extracts from Smilax Glabra and Na2SeO3 to chelating ability of lead ions and antioxidant effects[J]. Chin Arch Tradit Chin Med, 2018, 36(6):1392-5.
[4] Cui D, Yan C, Miao J, et al. Synthesis, characterization and antitumor properties of selenium nanoparticles coupling with ferulic acid[J]. Mater Sci Eng C Mater Biol Appl, 2018, 90:104-12.
[5] Sun X, Zhong Y, Luo H, et al. Selenium-containing polysaccharide-protein complex in Se-enriched ulva fasciata induces mitochondria-mediated apoptosis in A549 human lung cancer cells[J]. Mar Drugs, 2017, 15(7):pii:E215.
[6] Kim M S, Lee W S, Jin W. Trk B promotes breast cancer metastasis via suppression of runx3 and keap1 expression [J]. Mol Cells, 2015,12(10): 67-9.
[7] Na J, Jung J, Bang J, et al. Selenophosphate synthetase 1 and its role in redox homeostasis, defense and proliferation[J]. Free Radic Biol Med, 2018, 127: 190-7.
[8] 郭纪伟,武艳,金丹,等. 去甲斑蝥素调控YAP增强A549细胞对顺铂的敏感性[J]. 中国药理学通报,2018,34(5):717-23. Guo J W, Wu Y, Jin D, et al. Norcantharidin enhanced cisplatin sensitivity via YAP pathway in A549 cells[J]. Chin Pharmacol Bull, 2018, 34(5): 717-23.
[9] Li T, Xiang W, Li F, et al. Self-assembly regulated anticancer activity of platinum coordinated selenomethionine[J]. Biomaterials, 2018, 157:17-25.
[10] Chi S, Xu W, Liu J, et al. Effect of exogenous selenium on activities of antioxidant enzymes, cadmium accumulation and chemical forms of cadmium in tomatoes[J]. Int J Agric Biol, 2017, 19:1615-22.
[11] Ghorbel I, Elwej A, Chaabane M, et al. Selenium alleviates oxidative stress and lung damage induced by aluminum chloride in adult rats: biochemical and histological approach[J]. Biol Trace Elem Res, 2017, 176: 181-91.
[12] Zou B, Xiao G, Xu Y, et al. Persimmon vinegar polyphenols protect against hydrogen peroxide-induced cellular oxidative stress via Nrf2 signalling pathway[J]. Food Chem, 2018, 255: 23-30.
[13] 赵雨喆,姜京植,叶晶,等. 虎杖苷通过p38 MAPK /Nrf2/HO-1通路减轻小鼠哮喘模型气道炎症[J]. 中国药理学通报,2018,34(6):851-6. Zhao Y Z, Jiang J Z, Ye J, et al. Polydatin attenuates airway inflammation in asthmatic mouse model via p38 MAPK/Nrf 2/HO-1 pathway[J]. Chin Pharmacol Bull, 2018, 34(6): 851-6.
[14] 孟哲,腾帅,王琛,等. 积雪草酸通过Nrf2抗氧化通路抑制自发性高血压大鼠心肌纤维化[J]. 中国药理学通报,2018,34(8):1073-8. Meng Z, Teng S, Wang C, et al. Asiatic acid inhibits cardiac fibrosis via activating Nrf2-mediated antioxidant signaling pathway in spontaneous hypertensive rats[J]. Chin Pharmacol Bull, 2018, 34(8): 1073-8.