【作者】 王宁；

【导师】 杜智；

【作者基本信息】 天津医科大学 ， 外科学 普外（专业学位）， 2016， 硕士

【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。更多还原

【Abstract】 Enhancer of zeste homolog 2(EZH2) is a key componet of the polycomb repressive complex 2(PRC2), which has histone methyltransferase activity and can participate tri-methylation of H3K27 status. Epigenetic silencing induced by histone modification can be regarded as a risk factor for the inactivation of tumor-resistant genes. Corresponding to this, EZH2 can be recognized as a therapeutic target for cancer due to the phenomenon of the overexpression of EZH2 protein is quit commonly in various cancers. 3-deazaneplanocin(DZNep) is an inhibitor of S-adenosylhomocysteine hydrolase(SAHH), which could reduce the level of histone methylation and the status of methionine metabolism by indirectly inhibiting numberous methyltransferases activity. By now, DZNep has been employed as an anti-cancer drug which has proven effective in treatment for tumor suffers whose EZH2 protein is abnormal elevation. However, from essence, DZNep is not an inhibitor for EZH2 specifically; it is an inhibitor of SAHH as mentioned above. Hence, the mechiansm of the DZNep’s anti-cancer effects is controversial. Due to inhibiting SAHH is the most direct biochemical action of DZNep which resulte in the accumulating of the S-adenosylhomocysteine(SAH) and then inhibite many transmethylases activity including EZH2. The effects of the intracellular changes may influence various biologicale effects, i.e, methyl donor availability and the metabolic stress reaction. Therefore, we offer a hypothesis that the inhibiting of EZH2 should not be the only pathway for the mechanism of DZNep’s anti-cancer effects. Objective:We diucuss that the inhibiting of EZH2 should not be the only pathway for the mechanism of DZNep’s anti-cancer effects and whether DZNep could enhance the sensitivity to DNA-damaging stimuli by transforming the status of chromatin and then induces the cellular senescence. Methods:DZNep and GSK343 are different inhibitor of EZH2 molecule which is indirect and direct inhibitating EZH2, seperatly. Fistly, based on HepG2 cell model, cell growth curve was assayed after DZNep and GSK343 treatment by cell proliferation assays. Secondly, accoding to response to two drugs treatment, we analyzed the cell proliferation, death and morphology changes conditions, we hypothesized that the reason for DZNep can suppress tumor cell proliferation was by promoting cellular sescence. Thirdly, the senescence-like phenomena was analyzed by staining of senescence-associated β-galactosidasel. Forthly, based on analyzed cell cycle arrest, the expression of the cyclin-dependent kinase inhibitors, we recognized the signaling pathway which was influnced by DZNep treatment. Fifthly, employing cellular immune fluorescence analysis, the expression of p53 protein and its nuclear transfer ststus was identified to determine the activity of the p53. Finally, a penal of upstream signaling(γH2AX 、 ATM 、 CHK1 、 CHK2) in DNA damage response and senescence-associated inflammatory cytokine or chemokine secretion(IL6、IL8、IGFBP7) were assayed by RT-PCR, western blot and immunofluorescence analysis. Results:1. As an inhibitor of EZH2 directly, the death of HepG2 cell relied on GSK343 dose. However, the effect of DZNep inhibiting cell growth was not dose-dependent and DZNep could suppress the cell growth. Meanwhile, the accumulation of SAH could be found by use of HPLC qualitative analysis.2. The morphology changes of HepG2 cell after DZNep treatment was from round to flat. Besides, the intracellular particulate matter increased with DZNep treatment. Meanwhile, the staining of senescence-associated β-galactosidasel was green and this implied that the result of staining of SA--galAfter was positive. After all, the senescence-associated changes could be found in the HepG2 cell after DZNep treatment.3. The proportion of cells at G0/G1 phase was depressed and the proportion of cells increased from 11.01% to 28.72% after DZNep-treatment.DZNep retained the cell cycle at G2/M phase. With the corresponding, the expression of p21 which is a kind of cell cycle inhibitor was increased significantly.4. DZNep promoted the stabilization of p53 protein in a mild and chronic manner and induced p53 protein transfer into nuclear. Combined with our previous results, HepG2 cell senescence caused by DZNep treatment was mediated through p53-p21 pathway.5. A penal of upstream signaling in DDR, such as the expression of γH2AX、ATM、pCHK1 and pCHK2 was increased. Among this, the expression of ATM and pCHK1 at the day 24 was the highest and then began to attenuate gradually after 72 hours. The results implied that DNA damage response induced by DZNep treatment was early-effective of the drug.6. With HepG2 cell senescence induced by DZNep-treatment, inflammatory factors CXCR2, IGFBP7 and IL8 were dramatically increased. This implied that DDR promoted the cell senescence and these senescent cells further strengthen the effect of senescence by senescence-associated secretory phenotype.7. The results of microarray data confirmed that the top 10 pathways-related to the effect of DZNep treatment were histone isomer-associted, the result suggested that the effect of DZNep were relevant to chromatin state disturbance and remodelin. Meanwhile,the microarray data also pointed out that at least 3 known genes(ALDH3A, TRIM29, and TP53I3) closely associated with DNA repair were indentified to upregulated significantly in DZNep-treated cells. Conclusions:Aiming to explore the mechanism of DZNep anti-cancer effect, we found that the inhibiting of EZH2 should not be the only pathway for the mechanism of DZNep’s anti-cancer effects. HepG2 cellular senescence induced by DZNep was mediated through p53-p21 pathway and triggered by enhanced ATM activation related to chromatin changes. Our studies could provide some meaningful information as shown following:Firstly, our results suggested that similar with promote apoptosis of tumor cells, promoting tumor cell senescence was also a promising method to develop anti-tumor therapeutic;Secondly, employing the feature of DNA damage response induced by DZNep, along with in combination with radiotherapy and chemotherapy, seemed logical to improve the effectiveness of these traditional methods of cancer treatment.Finally, our studies implied that ATM could be activated induced by DZNep. However, which pathways of activation function was mediated by, i.e DNA breakage, ROS increase, chromatin state changes, etc., should be subject to further study.更多还原