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2020, v.45(24) 6012-6019

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阿司匹林联合长春瑞滨抑制非小细胞肺癌的作用及其机制研究
Effect and mechanism of aspirin combined with vinorelbine on non-small cell lung cancer

崔丽;邱亚楠;徐烨;滕玉鸥;郁彭;刘振;
CUI Li;QIU Ya-nan;XU Ye;TENG Yu-ou;YU Peng;LIU Zhen;School of Biological Engineering, Tianjin University of Science and Technology;

摘要(Abstract):

探究阿司匹林(aspirin)联合长春瑞滨(vinorelbine)对非小细胞肺癌细胞增殖、凋亡的影响及其潜在的作用机制。采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[(4,5-dimethythiazol-2,5-diphenyl)tetrazolium bromide,MTT]法检测阿司匹林和长春瑞滨对H460和A549细胞的细胞毒作用,计算其半数抑制浓度(IC_(50))和协同效果,结果显示,阿司匹林和长春瑞滨对H460和A549细胞的增殖抑制作用呈明显的浓度依赖性,在H460细胞中,其IC_(50)分别为1.553 mmol·L~(-1)和0.033μmol·L~(-1);在A549细胞中,阿司匹林的IC_(50)为1.70 mmol·L~(-1),长春瑞滨的IC_(50)大于20μmol·L~(-1);采用联合指数(combination index,CI)评价两药联合作用,发现阿司匹林和长春瑞滨的浓度比为100∶1时,两药联合对H460细胞增殖具有协同抑制作用(CI<1);当阿司匹林和长春瑞滨的浓度比为1∶10时,两药联合CI大部分小于1,对A549细胞具有一定的协同作用。采用克隆形成试验及4,6-联脒-2-苯基吲哚(4′,6-diamidino-2-phenylindole,DAPI)/碘化丙啶(propidium iodide,PI)染色试验验证两药联合对于肺癌细胞增殖的作用,结果显示,相较于阿司匹林单药组,联合组对肺癌细胞增殖抑制作用更强,克隆形成率为49.5%(P<0.05)。采用细胞凋亡、线粒体膜电位、活性氧及Western blot试验进一步探讨两药抑制细胞增殖的协同作用机制,结果显示两药联合后诱导肺癌细胞的凋亡率为52.8%,线粒体膜电位下降到33.1%,活性氧水平升高到73.3%,均显著高于单药处理组(P<0.05)。Western blot结果显示,与单药组相比,联合组能明显上调Bax,p53,cleaved caspase-3和细胞色素C(cytochrome C)蛋白的表达水平,同时下调抗凋亡蛋白Bcl-xL和Bcl-2的表达水平。以上研究结果表明,阿司匹林联合长春瑞滨能协同抑制H460细胞的增殖,其机制可能与线粒体途径诱导的细胞凋亡相关。
The study aimed to investigate the effect and mechanism of aspirin combined with vinorelbine on the proliferation and apoptosis of non-small cell lung cancer cells. 3-(4-dimethylthiazolyl-2)-2-diphenyltetrazolium bromide(MTT) was used to detect the cytotoxic effect of aspirin and vinorelbine on H460 and A549 cells, and half of inhibitory concentration(IC_(50)) value of drugs as well as synergistic effect were calculated. The results showed that both aspirin and vinorelbine inhibited the cancer cells proliferation by a concentration-dependent manner with IC_(50 )values of 1.553 mmol·L~(-1) and 0.033 μmol·L~(-1) in H460 cells, respectively. The IC_(50 )values of aspirin and vinorelbine were 1.70 mmol·L~(-1)and more than 20 μmol·L~(-1) in A549 cells. The combination index(CI) value was used to evaluate the combined effect of two drugs. Aspirin combined with vinorelbine had synergistic effects at the ratio of 100∶1 on H460 cells and 1∶10 on A549 cells(CI<1). Clone formation and 4′,6-diamidino-2-phenylindole(DAPI)/propidium iodide(PI) staining assays were used to verify the effect of the combination of two drugs on proliferation of H460 cells. Compared with the aspirin single group, the combination group had stronger inhibitory effect on the proliferation of H460 cells and the clone formation rate was 49.5%(P<0.05). Furthermore, apoptosis, mitochondrial membrane potential, reactive oxygen species and Western blot experiments were used to explore the synergistic mechanism of aspirin combined with vinorelbine in inhibiting cell proliferation. The results showed that the cancer cell apoptosis rate was 52.8%, the mitochondrial membrane potential was decreased to 33.1%, and the levels of reactive oxygen species was increased to 73.3% in combination group, which were significantly different from those of the single drug treatment groups(P<0.05). Western blot showed that combination group significantly up-regulated the expressions of Bax, p53, cleaved caspase-3 and cytochrome C, while down-regulated the expression of anti-apoptosis proteins such as Bcl-xL and Bcl-2 when compared with single groups. Our results suggested that aspirin combined with vinorelbine could synergistically inhibit the proliferation of H460 cells by inducing the cell apoptosis through the mitochondrial pathway.

关键词(KeyWords): 肺癌细胞;长春瑞滨;阿司匹林;细胞凋亡;线粒体
lung cancer cells;vinorelbine;aspirin;cell apoptosis;mitochondria

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81703014)

作者(Author): 崔丽;邱亚楠;徐烨;滕玉鸥;郁彭;刘振;
CUI Li;QIU Ya-nan;XU Ye;TENG Yu-ou;YU Peng;LIU Zhen;School of Biological Engineering, Tianjin University of Science and Technology;

Email:

DOI: 10.19540/j.cnki.cjcmm.20200924.401

参考文献(References):

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