苦荞茶多糖诱导人肺腺癌A549细胞凋亡的线粒体机制
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摘要
目的:从线粒体调控机制的角度探讨苦荞茶多糖(tartary buckwheat tea polysaccharide,TBTP)对人肺腺癌A549细胞凋亡的影响。方法:应用水提醇沉法结合超声辅助技术提取的TBTP处理A549细胞,分为空白对照组和不同质量浓度TBTP处理组。采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法和流式细胞术检测细胞增殖和凋亡水平,采用DHE和Mito SOX染色检测细胞和线粒体活性氧簇水平,采用JC-1染色检测线粒体膜电位变化,采用Western blot法检测凋亡相关蛋白Bcl-2相关X蛋白(Bcl-2 associated X protein,Bax)、Bcl-2、剪切型半胱氨酸蛋白酶(Caspase)-3、细胞色素c的水平。结果:TBTP呈剂量和时间依赖性抑制A549细胞增殖。TBTP处理A549细胞24 h可呈剂量依赖性地促进细胞凋亡。TBTP处理能够显著增加A549细胞和线粒体内活性氧水平,降低线粒体膜电位;同时,TBTP能够增加促凋亡蛋白Bax和剪切型Caspase-3的表达、抑制抗凋亡蛋白Bcl-2的表达,促进A549细胞线粒体细胞色素c外流。结论:TBTP能够促进活性氧簇生成、降低线粒体膜电位,进而抑制A549细胞增殖,促进细胞凋亡。
Objective:To investigate the effect of tartary buckwheat tea polysaccharide(TBTP) on the apoptosis of A549 human lung cancer cells and its underlying mechanisms.Methods:TBTP was obtained by water extraction.3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay was used to detect the influence of TBTP on the proliferation of A549 cells.Flow cytometry was applied to determine the effect of TBTP on cell apoptosis in A549 cells.The production of reactive oxygen species(ROS) was assessed by DHE and Mito SOX Red staining.The change of mitochondrial membrane potential was assessed by JC-1 staining.Meanwhile,Western blot analysis was used to evaluate the expression of Bcl-2-associated X protein(Bax),Bcl-2,cleaved caspase-3 and cytochrome c.Results:MTT assay showed a significant inhibitory effect of TBTP on A549 cells proliferation in a dose-and time-dependent manner.After 24 hours' cultivation,TBTP also promoted A549 cells apoptosis in a dose-dependent manner.TBTP treatment promoted mitochondrial ROS production and oxidative stress in A549 cells.The mitochondrial membrane potential of A549 cells also decreased after TBTP administration,accompanied with increased cytochrome c release from mitochondria.Furthermore,TBTP treatment up-regulated the expression of Bax and cleaved caspase-3,and decreased Bcl-2 expression.Conclusion:TBTP exerts a novel anti-proliferative effect through induction of mitochondria-mediated apoptosis in A549 cells.
Objective:To investigate the effect of tartary buckwheat tea polysaccharide(TBTP) on the apoptosis of A549 human lung cancer cells and its underlying mechanisms.Methods:TBTP was obtained by water extraction.3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT) assay was used to detect the influence of TBTP on the proliferation of A549 cells.Flow cytometry was applied to determine the effect of TBTP on cell apoptosis in A549 cells.The production of reactive oxygen species(ROS) was assessed by DHE and Mito SOX Red staining.The change of mitochondrial membrane potential was assessed by JC-1 staining.Meanwhile,Western blot analysis was used to evaluate the expression of Bcl-2-associated X protein(Bax),Bcl-2,cleaved caspase-3 and cytochrome c.Results:MTT assay showed a significant inhibitory effect of TBTP on A549 cells proliferation in a dose-and time-dependent manner.After 24 hours' cultivation,TBTP also promoted A549 cells apoptosis in a dose-dependent manner.TBTP treatment promoted mitochondrial ROS production and oxidative stress in A549 cells.The mitochondrial membrane potential of A549 cells also decreased after TBTP administration,accompanied with increased cytochrome c release from mitochondria.Furthermore,TBTP treatment up-regulated the expression of Bax and cleaved caspase-3,and decreased Bcl-2 expression.Conclusion:TBTP exerts a novel anti-proliferative effect through induction of mitochondria-mediated apoptosis in A549 cells.
引文
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[2]LU C L,ZHENG Q,SHEN Q,et al.Uncovering the relationship and mechanisms of tartary buckwheat(Fagopyrum tataricum)and type IIdiabetes,hypertension,and hyperlipidemia using a network pharmacology approach[J].PeerJ,2017,5(1):e4042.DOI:10.7717/peerj.4042.
[3]QIU J,LI Z G,QIN Y C,et al.Protective effect of tartary buckwheat on renal function in type 2 diabetics:a randomized controlled trial[J].Therapeutics&Clinical Risk Management,2016,12:1721-1727.DOI:10.2147/TCRM.S123008.
[4]QIU J,LIU Y,YUE Y,et al.Dietary tartary buckwheat intake attenuates insulin resistance and improves lipid profiles in patients with type 2 diabetes:a randomized controlled trial[J].Nutrition Research,2016,36(12):1-7.DOI:10.1016/j.nutres.2016.11.007.
[5]颜军,孙晓春,谢贞建,等.苦荞多糖的分离纯化及单糖组成测定[J].食品科学,2011,32(19):33-36.
[6]杨红燕,赵志壮,商庆超,等.苦荞茶多糖超声波提取工艺优化及组成分析[J].食品与发酵工业,2016,42(3):231-236.DOI:10.13995/j.cnki.11-1802/ts.201603041.
[7]李姗姗,崔晓东,李晨,等.苦荞多糖抗氧化及对HepG2细胞增殖抑制作用[J].食品研究与开发,2016,37(1):9-12.DOI:10.3969/j.issn.1005-6521.2016.01.003.
[8]WU S C,LEE B H.Buckwheat polysaccharide exerts antiproliferative effects in THP-1 human leukemia cells by inducing differentiation[J].Journal of Medicinal Food,2010,14(1/2):26-33.DOI:10.1089/jmf.2010.1252.
[9]石远凯,孙燕,丁翠敏,等.中国埃克替尼治疗非小细胞肺癌专家共识(2016年版)[J].中国肺癌杂志,2016,19(7):489-493.DOI:10.3779/j.issn.1009-3419.2016.07.12.
[10]CHEN W,ZHENG R,BAADE P D,et al.Cancer statistics in China,2015[J].CA:A Cancer Journal for Clinicians,2016,66(2):115-132.DOI:10.3322/caac.21338.
[11]孙艳婷,陈思成,李汪,等.PGRN基因沉默对人非小细胞肺癌A549细胞增殖及迁移的影响[J].中国病理生理杂志,2018,34(4):585-591.DOI:10.3969/j.issn.1000-4718.2018.04.002.
[12]ZUO H J,SHAN L,YAN C,et al.In vitro,and in vivo,evaluation of antitumor activity of Ligustrum robustum,a Chinese herbal tea[J].Chinese Journal of Integrative Medicine,2018,2018:1-6.DOI:10.1007/s11655-018-2983-5.
[13]DUAN Z,DENG J,DONG Y,et al.Anticancer effects of ginsenoside Rk3 on non-small cell lung cancer cells:in vitro and in vivo[J].Food&Function,2017,8(10):3723-3736.DOI:10.1039/c7fo00385d.
[14]WANG H B,TAO L D,NI T Y,et al.Anticancer efficacy of the ethyl acetate extract from the traditional Chinese medicine herb Celastrus orbiculatus against human gastric cancer[J].Journal of Ethnopharmacology,2017,205:147-157.DOI:10.1016/j.jep.2017.04.030.
[15]贾乔瑾,崔晓东,王转花.苦荞凝集素对结肠癌细胞增殖的抑制作用[J].中国生物化学与分子生物学报,2015(4):383-390.DOI:10.13865/j.cnki.cjbmb.2015.04.08.
[16]郭晓娜,姚惠源.苦荞麦蛋白质对乳腺癌细胞的作用机制[J].食品与生物技术学报,2011,30(1):55-59.
[17]李谣,周海媚,黄丹丹,等.苦荞抗氧化活性研究[J].中国酿造,2013,32(6):24-27.DOI:10.3969/j.issn.0254-5071.2013.06.007.
[18]SIKDAR S,MUKHERJEE A,GHOSH S,et al.Condurango glycoside-rich components stimulate DNA damage-induced cell cycle arrest and ROS-mediated caspase-3 dependent apoptosis through inhibition of cell-proliferation in lung cancer,in vitro and in vivo[J].Environmental Toxicology&Pharmacology,2014,37(1):300-314.DOI:10.1016/j.etap.2013.12.004.
[19]GU S Y,CHEN C Z,JIANG X J,et al.ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction underlie apoptosis induced by resveratrol and arsenic trioxide in A549 cells[J].Chemico-Biological Interactions,2016,245:100-109.DOI:10.1016/j.cbi.2016.01.005.
[20]LI J,WU D D,ZHANG J X,et al.Mitochondrial pathway mediated by reactive oxygen species involvement inα-hederin-induced apoptosis in hepatocellular carcinoma cells[J].World Journal of Gastroenterology,2018,24(17):1901-1910.DOI:10.3748/wjg.v24.i17.1901.
[21]SUN X,YU Z,LUO H,et al.Selenium-containing polysaccharideprotein complex in Se-enriched ulva fasciata induces mitochondriamediated apoptosis in A549 human lung cancer cells[J].Marine Drugs,2017,15(7):215-225.DOI:10.3390/md15070215.
[22]ZHENG P,CHEN Q,TIAN X,et al.DNA damage triggers tubular endoplasmic reticulum extension to promote apoptosis by facilitating ER-mitochondria signaling[J].Cell Research,2018,28(8):833-854.DOI:10.1038/s41422-018-0065-z.
[23]SHINY P J,MUKHERJEE A,CHANDRASEKARAN N.DNAdamage and mitochondria-mediated apoptosis of A549 lung carcinoma cells induced by biosynthesised silver and platinum nanoparticles[J].RSC Advances,2016,6(33):27775-27787.
[24]ASGHARI M H,ABDOLLAHI M,DE OLIVEIRA M R,et al.Areview of the protective role of melatonin during phosphine-induced cardiotoxicity:focus on mitochondrial dysfunction,oxidative stress and apoptosis[J].Journal of Pharmacy and Pharmacology,2017,69(3):236-243.DOI:10.1111/jphp.12682.
[25]易健,舒徐.活性氧对细胞凋亡和增殖的调控作用[J].基础医学与临床,2013,33(10):1127-1130.
[26]孙秀玉,刘立亚,吴宥熹,等.Bcl-2家族对线粒体质量控制的调控研究[J].中国药理学通报,2015,31(12):1633-1636.DOI:10.3969/j.issn.1001-1978.2015.12.002.
[27]POPGEORGIEV N,JABBOUR L,GILLET G.Subcellular localization and dynamics of the Bcl-2 family of proteins[J].Frontiers in Cell&Developmental Biology,2018,6:1-11.DOI:10.3389/fcell.2018.00013.
[28]GE L,SHAN K,WU S,et al.A novel polysaccharide from Sargassum integerrimum induces apoptosis in A549 cells and prevents angiogensis in vitro and in vivo[J].Scientific Reports,2016,6:26722.DOI:10.1038/srep26722.
[29]OPFERMAN J T,KOTHARI A.Anti-apoptotic Bcl-2 family members in development[J].Cell Death&Differentiation,2018,25(1):37-45.DOI:10.1038/cdd.2017.170.
[30]PENA-BLANCO A,GARCíA-SáEZ A J.Bax,Bak and beyondmitochondrial performance in apoptosis[J].The FEBS Journal,2018,285(3):416-431.DOI:10.1111/febs.14186.