Inhibitory effect of Euphorbia helioscopia on human triple-negative breast cancer MDA-MB-231 cells and its possible mechanism of apoptosis
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摘要: 目的 研究泽漆对三阴乳腺癌MDA-MB-231细胞的作用及其作用机制。 方法 用MTT法检测细胞活力；用荧光显微镜法测定MDA-MB-231细胞的活性氧（ROS）生成量；用流式细胞仪检测细胞的凋亡率；用TUNEL检测法检测细胞凋亡DNA碎片；用Western blot检测caspase-9、caspase-3、PARP等凋亡相关因子的水平变化。 结果 MTT试验显示泽漆提取物对MDA-MB-231细胞具有显著的抑制作用，但作用可被ROS抑制剂NAC及caspase抑制剂Z-VAD-FMK所消除；荧光显微镜检测显示泽漆提取物能显著提高ROS的生成；流式细胞仪检测显示泽漆提取物处理后，PI染色阳性细胞明显增加，但被NAC减弱。Caspase-9、caspase-3在提取物处理后均转为激活形式，PARP被剪切。TUNEL法显示，提取物处理后细胞凋亡碎片明显增多，而提前加入ROS抑制剂NAC和caspase抑制剂Z-VAD-FMK能使泽漆提取物诱导凋亡的DNA碎片明显减少。 结论 泽漆乙酸乙酯提取物可以有效抑制MDA-MB-231细胞的生长，诱导其凋亡，其作用机制可能与ROS过量生成所致的线粒体损伤途径有关。Abstract: Objective To investigate the effect of Euphorbia helioscopia on MDA-MB-231 cells and its mechanism. Methods The cell viability was detected by MTT assay. The production of ROS in MDA-MB-231 cells was measured by fluorescence microscopy. The apoptotic rate was detected by flow cytometry. Apoptosis DNA fragments were detected by TUNEL assay. Western blot was used to assess the expression of caspase-9, caspase-3 and PARP. Results MTT assay showed that the extract significantly inhibited the viability of MDA-MB-231 cells, which can be diminished by the ROS inhibitor NAC and the caspase inhibitor Z-VAD-FMK. The marked increase in the production of ROS induced by the extract was observed with fluorescence microscopy. Flow cytometry showed that the PI positive staining cells increased significantly after the treatment of the extract, but was diminished by NAC. Caspase-9 and caspase-3 were activated after the treatment of the extract while the PARP was cleaved. TUNEL showed that a significant increase in apoptotic DNA fragmentation induced by the extract, which can be diminished by NAC and Z-VAD-FMK. Conclusion Ethyl acetate extract inhibited the MDA-MB-231 cells and induced apoptosis. The mechanism may involve with the mitochondrial damage due to the excessive ROS.
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Key words:
- Euphorbia helioscopia /
- MDA-MB-231 cells /
- triple-negative breast cancer /
- ROS /
- mitochondrial pathway /
- apoptosis
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[1] Campbell RA, Bhat-nakshatri P, Patel NM, et al. Phosphatidylinositol 3-kinase/AKT-mediated activation of estrogen receptor alpha: a new model for anti-estrogen resistance[J]. J Biol Chem, 2001, 276(13): 9817-9824. [2] Hseu YC, Lee CC, Chen YC, et al. Antrodia salmonea in submerged culture exhibits antioxidant activities in vitro and protects human erythrocytes and low-density lipoproteins from oxidative modification[J]. Food Chem Toxicol, 2014, 66(4): 150-157. [3] Shah SP, Roth A, Goya R, et al. The clonal and mutational evolution spectrum of primary triple-negative breast cancers[J]. Nature, 2012, 486(7403): 395-399. [4] Cleator S, Heller W, Coombes RC. Triple-negative breast cancer: therapeutic options[J]. Lancet Oncol, 2007, 8(3): 235-244. [5] Wang K, Zhang C, Bao J, et al. Synergistic chemopreventive effects of curcumin and berberine on human breast cancer cells through induction of apoptosis and autophagic cell death[J]. Sci rep, 2016, 6: 26064. [6] Jeong HS, Choi HY, Lee ER, et al. Involvement of caspase-9 in autophagy-mediated cell survival pathway[J]. Biochim Biophys Acta, 2011, 1813(1): 80-90. [7] Green DR. Apoptotic pathways: paper wraps stone blunts scissors[J]. Cell, 2000, 102(1): 1-4. [8] 杨 莉, 陈海霞, 高文远. 泽漆化学成分及药理作用研究进展[J]. 中草药, 2007, 38(10): 1585-1589. [9] D'Anneo A, Carlisi D, Lauricella M, et al. Parthenolide generates reactive oxygen species and autophagy in MDA-MB231 cells. A soluble parthenolide analogue inhibits tumour growth and metastasis in a xenograft model of breast cancer[J]. Cell Death Dis, 2013, 4(10): e891. [10] Elumalai P, Gunadharini DN, Senthilkumar K, et al. Induction of apoptosis in human breast cancer cells by nimbolide through extrinsic and intrinsic pathway[J]. Toxicol Lett, 2012, 215(2): 131-142. [11] Hseu YC, Lee CC, Chen YC, et al. The anti-tumor activity of Antrodia salmonea in human promyelocytic leukemia (HL-60) cells is mediated via the induction of G(1) cell-cycle arrest and apoptosis in vitro or in vivo[J]. J Ethnopharmacol, 2014, 153(2): 499-510. [12] Mcilwain DR, Berger T, Mak TW. Caspase functions in cell death and disease[J]. Cold Spring HarbPerspect Biol, 2013, 5(4): a008656. -
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