木香烃内酯诱导人类非小细胞肺癌A549细胞凋亡的信号转导通路研究
发布时间：2019-02-11 13:45
【摘要】：目前肺癌仍是世界范围内一种严重威胁人类生命的肿瘤疾病,并且在我国该病的发病率有逐渐升高的趋势。尽管针对肺癌有许多治疗方法,但患者的5年存活率依然低于15%,且药物具有巨大的毒副作用,因此研发具有低毒高效抗肿瘤药物的需求仍是迫切的。木香烃内酯是从菊科木香属类植物中提取的一类倍半萜类化合物,能够体外抑制多种肿瘤细胞的生长,但对肺癌细胞的作用未见报道。本论文首先采用体外细胞生长抑制试验(MTT)检测木香烃内酯对不同肿瘤细胞生长抑制的作用,从而筛选出对药物最敏感的A549细胞；进而以A549细胞为研究对象,采用流式细胞术观察药物处理后细胞内ROS水平、膜电位以及细胞凋亡变化情况；此外还用激光共聚焦显微镜对细胞内钙离子以及膜电位变化情况进行观察；western blot (蛋白免疫印迹法)对细胞内内质网应激以及凋亡相关蛋白表达水平进行检测。具体得到的结果如下：1、MTT结果表明,木香烃内酯能体外抑制A549、HepG2、Hela细胞生长,且对肺癌细胞的生长抑制作用最明显,并且这种抑制作用表现一定的浓度和时间梯度依赖。2、以A549细胞为模型,应用流式细胞技术,通过Annexin V/PI双染细胞后观察不同浓度的木香烃内酯处理细胞24h的凋亡情况。结果表明,细胞凋亡率随着药物浓度的升高而增加,呈现出一定的浓度梯度依赖性。3、木香烃内酯处理A549细胞后发现,能够显著提升细胞内ROS的水平,并诱导内质网发生应激,内质网应激蛋白GRP78, IRE1α, CHOP等蛋白水平随着木香烃内酯浓度的升高而增加,这一过程伴随着胞质中钙离子水平的升高。通过RNA干扰技术抑制IRE1α表达可以使木香烃内酯诱导的细胞凋亡率下降,证明了内质网应激在木香烃内酯诱导凋亡中的作用。进一步研究表明通过IRE1α-ASK1-JNK通路磷酸化Bcl-2蛋白,使其失去抗凋亡作用,改变了线粒体膜通透性。应用流式细胞技术,通过JC-1染色发现药物处理后线粒体膜电位显著降低。抗氧化剂NAC能够恢复细胞活性、线粒体膜电位以及有效抑制细胞凋亡的发生。表明ROS是木香烃内酯诱导A549细胞发生凋亡的上游信号。4、不同的药物浓度处理A549细胞后对caspase活性进行检测,结果表明木香烃内酯能够激活caspase3和caspase9。caspase抑制剂Z-VAD-FMK能够有效恢复细胞生长活性；western blot检测显示,木香烃内酯上调了Bax、下调了Bcl-2的表达量,并且促使细胞色素c从线粒体释放,激活了caspase3,最终切割PARP,导致细胞发生线粒体途径的凋亡。本文首次阐明木香烃内酯通过内质网氧化应激介导IRE1α-JNK的激活,导致Bcl-2磷酸化失去抗凋亡作用,诱导A549细胞发生线粒体途径凋亡。研究结果进一步揭示木香烃内酯抗肿瘤机理,为将来木香烃内酯能够作为临床新型抗肿瘤药物提供相关的理论依据
[Abstract]:At present, lung cancer is still a serious threat to human life in the world, and the incidence of lung cancer is gradually increasing in China. Although there are many treatments for lung cancer, the 5-year survival rate of patients is still lower than 15%, and the drug has great toxicity and side effects, so it is urgent to develop anti-tumor drugs with low toxicity and high efficiency. Xylenolactone is a kind of sesquiterpenoids extracted from Compositae, which can inhibit the growth of many kinds of tumor cells in vitro, but the effect on lung cancer cells has not been reported. In this paper, in vitro cell growth inhibition test (MTT) was used to detect the inhibitory effect of xylinolactone on different tumor cells, so as to select the most sensitive A549 cells. The changes of ROS level, membrane potential and apoptosis of A549 cells were observed by flow cytometry. The changes of intracellular Ca ~ (2 +) and membrane potential were observed by laser confocal microscopy (LSCM). The changes of intracellular endoplasmic reticulum (ER) stress and the expression of apoptosis-related proteins were detected by; western blot (Western blot. The results were as follows: 1 MTT results showed that xylinolactone could inhibit the growth of A549 HepG2Hela cells in vitro, and had the most obvious inhibitory effect on the growth of lung cancer cells. The inhibition showed a certain concentration and time gradient dependence. 2. A549 cells were used as a model. Flow cytometry was used to observe the apoptosis of A549 cells treated with different concentrations of xylinolactone for 24 hours by Annexin V/PI double staining. The results showed that the apoptosis rate of A549 cells increased with the increase of drug concentration, and showed a certain concentration gradient dependence. 3. After treated with xylinolactone, it was found that the level of ROS in A549 cells could be significantly increased by xylinolactone. Endoplasmic reticulum stress proteins (GRP78, IRE1 伪, CHOP, etc.) increased with the increase of xylinolactone concentration, which was accompanied by the increase of calcium level in the cytoplasm. Inhibition of IRE1 伪 expression by RNA interference technique can decrease the apoptosis rate induced by xylinolactone, which proves the role of endoplasmic reticulum stress in the apoptosis induced by xylinolactone. Further studies showed that the phosphorylated Bcl-2 protein was phosphorylated through the IRE1 伪-ASK1-JNK pathway, which caused it to lose its anti-apoptotic effect and change the permeability of mitochondrial membrane. Flow cytometry and JC-1 staining showed that mitochondrial membrane potential was significantly decreased after drug treatment. Antioxidant NAC can restore cell activity, mitochondrial membrane potential and inhibit apoptosis. The results showed that ROS was the upstream signal of A549 cells apoptosis induced by xylinolactone. 4. The activity of caspase was detected after different concentrations of drugs were used to treat A549 cells. The results showed that xylinolactone could activate caspase3 and caspase9.caspase inhibitor Z-VAD-FMK could effectively restore cell growth activity. Western blot analysis showed that xylinolactone upregulated Bax, down-regulated the expression of Bcl-2 and promoted the release of cytochrome c from mitochondria, which activated caspase3, to cut PARP, and lead to apoptosis of mitochondria pathway. It is the first time to elucidate the activation of IRE1 伪-JNK mediated by xylinolactone through endoplasmic reticulum oxidative stress, which results in Bcl-2 phosphorylation losing its antiapoptotic effect and inducing apoptosis of mitochondria pathway in A549 cells. The results further revealed the antitumor mechanism of xylenolactone, and provided the relevant theoretical basis for the future use of xylol lactone as a new clinical antitumor drug.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2
本文编号：2419784
[Abstract]:At present, lung cancer is still a serious threat to human life in the world, and the incidence of lung cancer is gradually increasing in China. Although there are many treatments for lung cancer, the 5-year survival rate of patients is still lower than 15%, and the drug has great toxicity and side effects, so it is urgent to develop anti-tumor drugs with low toxicity and high efficiency. Xylenolactone is a kind of sesquiterpenoids extracted from Compositae, which can inhibit the growth of many kinds of tumor cells in vitro, but the effect on lung cancer cells has not been reported. In this paper, in vitro cell growth inhibition test (MTT) was used to detect the inhibitory effect of xylinolactone on different tumor cells, so as to select the most sensitive A549 cells. The changes of ROS level, membrane potential and apoptosis of A549 cells were observed by flow cytometry. The changes of intracellular Ca ~ (2 +) and membrane potential were observed by laser confocal microscopy (LSCM). The changes of intracellular endoplasmic reticulum (ER) stress and the expression of apoptosis-related proteins were detected by; western blot (Western blot. The results were as follows: 1 MTT results showed that xylinolactone could inhibit the growth of A549 HepG2Hela cells in vitro, and had the most obvious inhibitory effect on the growth of lung cancer cells. The inhibition showed a certain concentration and time gradient dependence. 2. A549 cells were used as a model. Flow cytometry was used to observe the apoptosis of A549 cells treated with different concentrations of xylinolactone for 24 hours by Annexin V/PI double staining. The results showed that the apoptosis rate of A549 cells increased with the increase of drug concentration, and showed a certain concentration gradient dependence. 3. After treated with xylinolactone, it was found that the level of ROS in A549 cells could be significantly increased by xylinolactone. Endoplasmic reticulum stress proteins (GRP78, IRE1 伪, CHOP, etc.) increased with the increase of xylinolactone concentration, which was accompanied by the increase of calcium level in the cytoplasm. Inhibition of IRE1 伪 expression by RNA interference technique can decrease the apoptosis rate induced by xylinolactone, which proves the role of endoplasmic reticulum stress in the apoptosis induced by xylinolactone. Further studies showed that the phosphorylated Bcl-2 protein was phosphorylated through the IRE1 伪-ASK1-JNK pathway, which caused it to lose its anti-apoptotic effect and change the permeability of mitochondrial membrane. Flow cytometry and JC-1 staining showed that mitochondrial membrane potential was significantly decreased after drug treatment. Antioxidant NAC can restore cell activity, mitochondrial membrane potential and inhibit apoptosis. The results showed that ROS was the upstream signal of A549 cells apoptosis induced by xylinolactone. 4. The activity of caspase was detected after different concentrations of drugs were used to treat A549 cells. The results showed that xylinolactone could activate caspase3 and caspase9.caspase inhibitor Z-VAD-FMK could effectively restore cell growth activity. Western blot analysis showed that xylinolactone upregulated Bax, down-regulated the expression of Bcl-2 and promoted the release of cytochrome c from mitochondria, which activated caspase3, to cut PARP, and lead to apoptosis of mitochondria pathway. It is the first time to elucidate the activation of IRE1 伪-JNK mediated by xylinolactone through endoplasmic reticulum oxidative stress, which results in Bcl-2 phosphorylation losing its antiapoptotic effect and inducing apoptosis of mitochondria pathway in A549 cells. The results further revealed the antitumor mechanism of xylenolactone, and provided the relevant theoretical basis for the future use of xylol lactone as a new clinical antitumor drug.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R734.2
【参考文献】
相关期刊论文 前2条
1 王晓庆,梁中琴,顾振纶;姜黄素抗肿瘤作用机制研究进展[J];中草药;2004年03期
2 Eckart Laack,Dieter Kurt Hossfeld,廖永德;非小细胞性肺癌的治疗[J];The Chinese-German Journal of Clinical Oncology;2000年06期
，本文编号：2419784
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2419784.html
