DZNep通过DNA损伤反应诱导肝癌细胞衰老
发布时间：2018-08-02 08:44
【摘要】：EZH2(enhancer of zeste homolog 2)是多梳抑制复合物2(polycomb repressive complex 2,PRC2)的核心成员,具有组蛋白甲基转移酶活性,可以催化组蛋白3第27位赖氨基酸上的三甲基化(H3K27me3)。这一组蛋白修饰介导的基因表观遗传沉默,是许多肿瘤抑制基因失活的方式之一;与此相应,EZH2在多种癌症的发生发展中是过度表达的,因此被看作为一个癌症治疗的靶点。DZNep是第一个通过药物筛选而被报道的EZH2小分子抑制剂,目前已有很多研究将这一药物应用于EZH2异常增高的肿瘤细胞或癌症病人,并已取得一定的抗肿瘤效果。然而DZNep本质上是S-腺苷高半胱氨酸水解酶(S-adenosyl homocysteine hydrolase,SAHH)的抑制剂,理论上并不是一种特异性EZH2的抑制剂。因此,关于DZNep的抗肿瘤机制是否仅仅是通过抑制EZH2活性、逆转特定抑癌基因的组蛋白甲基化而使其恢复表达,对此目前的研究报道还存在相互矛盾的结果或解释。由于DZNep最直接的生化作用是抑制SAHH,导致细胞内甲基转移反应的共同产物S-腺苷高半胱氨酸(S-adenosyl homocysteine,SAH)积累,从而间接地抑制包括EZH2在内的多种甲基转移酶活性;这些作用的效果可能涉及细胞内一碳单位的循环、甲基供体的可获得性以及与代谢有关的应激反应等多种生物学效应。因此,我们推测DZNep的抗肿瘤效果可能也不是单一的。进一步阐明DZNep的抗肿瘤机制,可以为这类药物的临床应用提供理论基础。【目的】探讨DZNep可能非依赖于EZH2的抗肿瘤生物功能及是否可以通过改变细胞的染色质结构,进而增强对DNA损伤反应的敏感性,诱导细胞衰老,进而发挥肿瘤抑制作用。【方法】以HepG2细胞株为模型,首先比较两种分别间接和直接抑制EZH2的小分子药物作用效果的差异,根据细胞在增殖、死亡、形态等方面的表现,推测DZNep抑制肿瘤细胞增殖主要是通过促进细胞的衰老,并通过β-半乳糖苷酶染色实验加以证实;根据细胞周期阻滞的时期、细胞周期抑制蛋白的表达,确定DZNep促进细胞衰老的信号途径;通过细胞免疫荧光分析p53的表达量与核内转移确定其激活状态;最后通过分析DNA损伤反应(DNA Damage Response,DDR)通路的上游信号与炎症相关因子的表达,确定dznep促进的细胞衰老是通过ddr信号途径引发、并通过炎症因子得以维持和增强。【结果】1.ezh2的直接抑制剂gsk343对hepg2的作用主要引起剂量依赖型的细胞死亡,而dznep的效应则是抑制细胞生长,同时高效液相定性分析可见sah在细胞内积累;2.dznep处理引起细胞形态变为扁平、胞内颗粒物增加等衰老样改变,且sa-b-gal染色呈阳性绿色;3.dznep使g2/m期细胞比例从11.01%提高到28.72%,提示细胞周期阻滞于g2/m期;与此相应,细胞周期抑制相关因子p21表达升高4.dznep可促进p53蛋白在细胞内稳定性增加并向核内转移,与上一结果相结合,表明dznep导致的细胞衰老是通过p53-p21途径;5.在ddr的上游信号中,γh2ax、atm、pchk1和pchk2表达增强,其中atm、pchk1在24小时内表达明显增强;而ezh2则在72小时后表达逐渐减弱,表明dznep诱发的ddr是其药物作用的早期效应;6.伴随dznep导致的细胞衰老,炎性因子cxcr2、igfbp7和il8基因的转录水平明显提高,表明dznep通过ddr途径促进细胞衰老,而衰老的细胞又通过衰老相关分泌表型(senescence-associatedsecretoryphenotype,sasp)进一步强化了细胞的衰老效应。7.通过表达谱芯片分析表明,在10个与dznep效应最相关的信号通路中,前两个均为组蛋白异构体相关基因,提示dznep的效应可能与染色质状态的扰动与重构有关;同时芯片分析还初步鉴定了三个可能通过参与dna修复而降低抗癌药物作用效果的基因(aldh3a,trim29,和tp53i3)。【结论】本研究通过探讨dznep非依赖于ezh2的生物学功能,发现它可通过激活atm信号途径来诱发dna损伤反应,并通过p53-p21通路引起细胞衰老。我们的这一发现具有以下两点意义:首先,我们的研究表明,与促进肿瘤细胞凋亡一样,促进肿瘤细胞的衰老同样是一条值得探索和开发的抗肿瘤途径;其次,可以利用dznep能够诱发dna损伤反应这一特性,与放疗、化疗手段相结合,提高这些传统的癌症治疗手段的效果。最后,本研究表明DZNep可以激活ATM,然而这种激活作用究竟是通过何种具体方式(DNA断裂、ROS增加、染色质状态改变等),还有待于进一步深入研究。
[Abstract]:EZH2 (enhancer of zeste homolog 2) is the core member of the multi comb inhibitory complex 2 (Polycomb repressive complex 2, PRC2), with the histone methyltransferase activity, which can catalyze the tri methylation (H3K27me3) of the histone 3 twenty-seventh bits of the Lycic amino acid. This histone trimming mediated gene epigenetic silencing is a number of tumor suppressor As one of the ways of inactivation, EZH2 is overexpressed in the development of a variety of cancers, so it is seen as a target for cancer treatment,.DZNep, the first EZH2 small molecule inhibitor reported by drug screening. Many studies have now applied this drug to abnormal EZH2 tumor cells or cancer. DZNep is essentially an inhibitor of S- adenosine homocysteine hydrolase (S-adenosyl homocysteine hydrolase, SAHH), but it is not a specific inhibitor of EZH2 in theory. Therefore, the anti tumor mechanism of DZNep is only by inhibiting the activity of EZH2 to reverse a specific tumor suppressor. There are conflicting results or explanations for the methylation of histones in the gene, and there are conflicting results or explanations. The most direct biochemical action of DZNep is the accumulation of S- adenosine homocysteine (S-adenosyl homocysteine, SAH), a common product of the intracellular methyl transfer reaction, which indirectly inhibits the inclusion of the SAHH. Several methyltransferase activities, such as EZH2, may be involved in a variety of biological effects, such as the cycle of one carbon unit in the cell, the availability of the methyl donor and the stress response related to metabolism. Therefore, we speculate that the antitumor effect of DZNep may not be single. Further elucidates the anti-tumor mechanism of DZNep, [Objective] to provide a theoretical basis for the clinical application of this kind of drug. [Objective] to explore the anti tumor biological function of DZNep which may not depend on EZH2 and whether it can change the chromatin structure of the cells, and then enhance the sensitivity of the DNA damage response, induce cell senescence, and then release the tumor suppressor effect. [method] HepG2 cell line For the model, we first compare the differences in the effect of two small molecular drugs, which are indirect and direct inhibition of EZH2. According to the cell proliferation, death, and morphology, it is speculated that DZNep inhibits the proliferation of the tumor cells mainly by promoting cell senescence and by beta galactosidase staining, according to the cell cycle resistance. In the period of stagnation, the expression of cell cycle inhibits protein to determine the signal pathway for DZNep to promote cell senescence; to determine the activation state of p53 by cell immunofluorescence analysis and intra nuclear transfer; finally, the expression of the upstream signal of the DNA damage response (DNA Damage Response, DDR) pathway and the expression of inflammation related factors is determined to determine DZNep promotion. Cellular senescence was triggered by DDR signaling pathway and maintained and enhanced through inflammatory factors. [results] the effect of 1.ezh2's direct inhibitor, gsk343, on HepG2 was mainly caused by dose dependent cell death, while DZNep effect was the inhibition of cell growth. At the same time, high performance liquid phase qualitative analysis showed that SAH was accumulated in cells; 2.dzn EP treatment caused cell morphology to become flat, intracellular particles increased and other senescence like changes, and sa-b-gal staining was positive green; 3.dznep increased the proportion of g2/m cells from 11.01% to 28.72%, suggesting that cell cycle arrest in g2/m phase; correspondingly, the increase of 4.dznep in cell cycle inhibition related to p21 expression could promote the stability of p53 protein in cell. Qualitative increase and transfer to the nucleus, combined with the previous results, indicating that the cell senescence caused by DZNep is through the p53-p21 pathway; 5. in the upstream signal of DDR, the expression of gamma, ATM, pchk1 and pchk2 is enhanced, in which ATM and pchk1 are markedly enhanced in 24 hours, while EZH2 decreases gradually after 72 hours, indicating DZNep induced DDR is its drug. The early effect of the action of the substance; 6. with the cell senescence associated with DZNep, the transcriptional level of the inflammatory factors CXCR2, IGFBP7 and IL8 genes is obviously improved, indicating that DZNep promotes cell senescence through the DDR pathway, and the senescent cells further strengthen the cell senescence through the senescence related secretory phenotype (senescence-associatedsecretoryphenotype, SASP). Effect.7. through expression spectrum chip analysis showed that the first two of the 10 signal pathways most related to the DZNep effect were histone isomer related genes, suggesting that the effect of DZNep may be related to the disturbance and remodeling of chromatin state; meanwhile, the chip analysis also preliminarily identified three anticancer drugs by participating in DNA repair. Gene (aldh3a, trim29, and tp53i3). [Conclusion] this study shows that DZNep is not dependent on the biological function of EZH2, and it is found that it can induce the DNA damage response by activating the ATM signal pathway and causing cell senescence through the p53-p21 pathway. Our findings have two points of significance: first, our research shows Like promoting tumor cell apoptosis, promoting tumor cell senescence is also an antitumor approach that is worth exploring and developing. Secondly, DZNep can induce DNA damage response, combined with radiotherapy and chemotherapy, to improve the effect of these traditional cancer treatments. Finally, this study shows that DZNep can be stimulated. Active ATM, however, remains to be further studied in what specific ways this activation takes (DNA breakage, increased ROS, altered chromatin status, etc.).
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7
本文编号：2158865
[Abstract]:EZH2 (enhancer of zeste homolog 2) is the core member of the multi comb inhibitory complex 2 (Polycomb repressive complex 2, PRC2), with the histone methyltransferase activity, which can catalyze the tri methylation (H3K27me3) of the histone 3 twenty-seventh bits of the Lycic amino acid. This histone trimming mediated gene epigenetic silencing is a number of tumor suppressor As one of the ways of inactivation, EZH2 is overexpressed in the development of a variety of cancers, so it is seen as a target for cancer treatment,.DZNep, the first EZH2 small molecule inhibitor reported by drug screening. Many studies have now applied this drug to abnormal EZH2 tumor cells or cancer. DZNep is essentially an inhibitor of S- adenosine homocysteine hydrolase (S-adenosyl homocysteine hydrolase, SAHH), but it is not a specific inhibitor of EZH2 in theory. Therefore, the anti tumor mechanism of DZNep is only by inhibiting the activity of EZH2 to reverse a specific tumor suppressor. There are conflicting results or explanations for the methylation of histones in the gene, and there are conflicting results or explanations. The most direct biochemical action of DZNep is the accumulation of S- adenosine homocysteine (S-adenosyl homocysteine, SAH), a common product of the intracellular methyl transfer reaction, which indirectly inhibits the inclusion of the SAHH. Several methyltransferase activities, such as EZH2, may be involved in a variety of biological effects, such as the cycle of one carbon unit in the cell, the availability of the methyl donor and the stress response related to metabolism. Therefore, we speculate that the antitumor effect of DZNep may not be single. Further elucidates the anti-tumor mechanism of DZNep, [Objective] to provide a theoretical basis for the clinical application of this kind of drug. [Objective] to explore the anti tumor biological function of DZNep which may not depend on EZH2 and whether it can change the chromatin structure of the cells, and then enhance the sensitivity of the DNA damage response, induce cell senescence, and then release the tumor suppressor effect. [method] HepG2 cell line For the model, we first compare the differences in the effect of two small molecular drugs, which are indirect and direct inhibition of EZH2. According to the cell proliferation, death, and morphology, it is speculated that DZNep inhibits the proliferation of the tumor cells mainly by promoting cell senescence and by beta galactosidase staining, according to the cell cycle resistance. In the period of stagnation, the expression of cell cycle inhibits protein to determine the signal pathway for DZNep to promote cell senescence; to determine the activation state of p53 by cell immunofluorescence analysis and intra nuclear transfer; finally, the expression of the upstream signal of the DNA damage response (DNA Damage Response, DDR) pathway and the expression of inflammation related factors is determined to determine DZNep promotion. Cellular senescence was triggered by DDR signaling pathway and maintained and enhanced through inflammatory factors. [results] the effect of 1.ezh2's direct inhibitor, gsk343, on HepG2 was mainly caused by dose dependent cell death, while DZNep effect was the inhibition of cell growth. At the same time, high performance liquid phase qualitative analysis showed that SAH was accumulated in cells; 2.dzn EP treatment caused cell morphology to become flat, intracellular particles increased and other senescence like changes, and sa-b-gal staining was positive green; 3.dznep increased the proportion of g2/m cells from 11.01% to 28.72%, suggesting that cell cycle arrest in g2/m phase; correspondingly, the increase of 4.dznep in cell cycle inhibition related to p21 expression could promote the stability of p53 protein in cell. Qualitative increase and transfer to the nucleus, combined with the previous results, indicating that the cell senescence caused by DZNep is through the p53-p21 pathway; 5. in the upstream signal of DDR, the expression of gamma, ATM, pchk1 and pchk2 is enhanced, in which ATM and pchk1 are markedly enhanced in 24 hours, while EZH2 decreases gradually after 72 hours, indicating DZNep induced DDR is its drug. The early effect of the action of the substance; 6. with the cell senescence associated with DZNep, the transcriptional level of the inflammatory factors CXCR2, IGFBP7 and IL8 genes is obviously improved, indicating that DZNep promotes cell senescence through the DDR pathway, and the senescent cells further strengthen the cell senescence through the senescence related secretory phenotype (senescence-associatedsecretoryphenotype, SASP). Effect.7. through expression spectrum chip analysis showed that the first two of the 10 signal pathways most related to the DZNep effect were histone isomer related genes, suggesting that the effect of DZNep may be related to the disturbance and remodeling of chromatin state; meanwhile, the chip analysis also preliminarily identified three anticancer drugs by participating in DNA repair. Gene (aldh3a, trim29, and tp53i3). [Conclusion] this study shows that DZNep is not dependent on the biological function of EZH2, and it is found that it can induce the DNA damage response by activating the ATM signal pathway and causing cell senescence through the p53-p21 pathway. Our findings have two points of significance: first, our research shows Like promoting tumor cell apoptosis, promoting tumor cell senescence is also an antitumor approach that is worth exploring and developing. Secondly, DZNep can induce DNA damage response, combined with radiotherapy and chemotherapy, to improve the effect of these traditional cancer treatments. Finally, this study shows that DZNep can be stimulated. Active ATM, however, remains to be further studied in what specific ways this activation takes (DNA breakage, increased ROS, altered chromatin status, etc.).
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.7
【参考文献】
相关期刊论文 前3条
1 曹相玫;郭东更;谷雨;蒋丽娜;李青;;EZH2在原发性肝癌发展中的作用及临床病理意义[J];现代医药卫生;2012年23期
2 ;Overexpression of polo-like kinase1 predicts a poor prognosis in hepatocellular carcinoma patients[J];World Journal of Gastroenterology;2009年33期
3 ;The prognostic molecular markers in hepatocellular carcinoma[J];World Journal of Gastroenterology;2002年03期
，本文编号：2158865
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2158865.html
