DZNep通過(guò)DNA損傷反應(yīng)誘導(dǎo)肝癌細(xì)胞衰老
發(fā)布時(shí)間：2018-08-02 08:44
【摘要】：EZH2(enhancer of zeste homolog 2)是多梳抑制復(fù)合物2(polycomb repressive complex 2,PRC2)的核心成員,具有組蛋白甲基轉(zhuǎn)移酶活性,可以催化組蛋白3第27位賴(lài)氨基酸上的三甲基化(H3K27me3)。這一組蛋白修飾介導(dǎo)的基因表觀遺傳沉默,是許多腫瘤抑制基因失活的方式之一;與此相應(yīng),EZH2在多種癌癥的發(fā)生發(fā)展中是過(guò)度表達(dá)的,因此被看作為一個(gè)癌癥治療的靶點(diǎn)。DZNep是第一個(gè)通過(guò)藥物篩選而被報(bào)道的EZH2小分子抑制劑,目前已有很多研究將這一藥物應(yīng)用于EZH2異常增高的腫瘤細(xì)胞或癌癥病人,并已取得一定的抗腫瘤效果。然而DZNep本質(zhì)上是S-腺苷高半胱氨酸水解酶(S-adenosyl homocysteine hydrolase,SAHH)的抑制劑,理論上并不是一種特異性EZH2的抑制劑。因此,關(guān)于DZNep的抗腫瘤機(jī)制是否僅僅是通過(guò)抑制EZH2活性、逆轉(zhuǎn)特定抑癌基因的組蛋白甲基化而使其恢復(fù)表達(dá),對(duì)此目前的研究報(bào)道還存在相互矛盾的結(jié)果或解釋。由于DZNep最直接的生化作用是抑制SAHH,導(dǎo)致細(xì)胞內(nèi)甲基轉(zhuǎn)移反應(yīng)的共同產(chǎn)物S-腺苷高半胱氨酸(S-adenosyl homocysteine,SAH)積累,從而間接地抑制包括EZH2在內(nèi)的多種甲基轉(zhuǎn)移酶活性;這些作用的效果可能涉及細(xì)胞內(nèi)一碳單位的循環(huán)、甲基供體的可獲得性以及與代謝有關(guān)的應(yīng)激反應(yīng)等多種生物學(xué)效應(yīng)。因此,我們推測(cè)DZNep的抗腫瘤效果可能也不是單一的。進(jìn)一步闡明DZNep的抗腫瘤機(jī)制,可以為這類(lèi)藥物的臨床應(yīng)用提供理論基礎(chǔ)。【目的】探討DZNep可能非依賴(lài)于EZH2的抗腫瘤生物功能及是否可以通過(guò)改變細(xì)胞的染色質(zhì)結(jié)構(gòu),進(jìn)而增強(qiáng)對(duì)DNA損傷反應(yīng)的敏感性,誘導(dǎo)細(xì)胞衰老,進(jìn)而發(fā)揮腫瘤抑制作用。【方法】以HepG2細(xì)胞株為模型,首先比較兩種分別間接和直接抑制EZH2的小分子藥物作用效果的差異,根據(jù)細(xì)胞在增殖、死亡、形態(tài)等方面的表現(xiàn),推測(cè)DZNep抑制腫瘤細(xì)胞增殖主要是通過(guò)促進(jìn)細(xì)胞的衰老,并通過(guò)β-半乳糖苷酶染色實(shí)驗(yàn)加以證實(shí);根據(jù)細(xì)胞周期阻滯的時(shí)期、細(xì)胞周期抑制蛋白的表達(dá),確定DZNep促進(jìn)細(xì)胞衰老的信號(hào)途徑;通過(guò)細(xì)胞免疫熒光分析p53的表達(dá)量與核內(nèi)轉(zhuǎn)移確定其激活狀態(tài);最后通過(guò)分析DNA損傷反應(yīng)(DNA Damage Response,DDR)通路的上游信號(hào)與炎癥相關(guān)因子的表達(dá),確定dznep促進(jìn)的細(xì)胞衰老是通過(guò)ddr信號(hào)途徑引發(fā)、并通過(guò)炎癥因子得以維持和增強(qiáng)。【結(jié)果】1.ezh2的直接抑制劑gsk343對(duì)hepg2的作用主要引起劑量依賴(lài)型的細(xì)胞死亡,而dznep的效應(yīng)則是抑制細(xì)胞生長(zhǎng),同時(shí)高效液相定性分析可見(jiàn)sah在細(xì)胞內(nèi)積累;2.dznep處理引起細(xì)胞形態(tài)變?yōu)楸馄�、胞�?nèi)顆粒物增加等衰老樣改變,且sa-b-gal染色呈陽(yáng)性綠色;3.dznep使g2/m期細(xì)胞比例從11.01%提高到28.72%,提示細(xì)胞周期阻滯于g2/m期;與此相應(yīng),細(xì)胞周期抑制相關(guān)因子p21表達(dá)升高4.dznep可促進(jìn)p53蛋白在細(xì)胞內(nèi)穩(wěn)定性增加并向核內(nèi)轉(zhuǎn)移,與上一結(jié)果相結(jié)合,表明dznep導(dǎo)致的細(xì)胞衰老是通過(guò)p53-p21途徑;5.在ddr的上游信號(hào)中,γh2ax、atm、pchk1和pchk2表達(dá)增強(qiáng),其中atm、pchk1在24小時(shí)內(nèi)表達(dá)明顯增強(qiáng);而ezh2則在72小時(shí)后表達(dá)逐漸減弱,表明dznep誘發(fā)的ddr是其藥物作用的早期效應(yīng);6.伴隨dznep導(dǎo)致的細(xì)胞衰老,炎性因子cxcr2、igfbp7和il8基因的轉(zhuǎn)錄水平明顯提高,表明dznep通過(guò)ddr途徑促進(jìn)細(xì)胞衰老,而衰老的細(xì)胞又通過(guò)衰老相關(guān)分泌表型(senescence-associatedsecretoryphenotype,sasp)進(jìn)一步強(qiáng)化了細(xì)胞的衰老效應(yīng)。7.通過(guò)表達(dá)譜芯片分析表明,在10個(gè)與dznep效應(yīng)最相關(guān)的信號(hào)通路中,前兩個(gè)均為組蛋白異構(gòu)體相關(guān)基因,提示dznep的效應(yīng)可能與染色質(zhì)狀態(tài)的擾動(dòng)與重構(gòu)有關(guān);同時(shí)芯片分析還初步鑒定了三個(gè)可能通過(guò)參與dna修復(fù)而降低抗癌藥物作用效果的基因(aldh3a,trim29,和tp53i3)。【結(jié)論】本研究通過(guò)探討dznep非依賴(lài)于ezh2的生物學(xué)功能,發(fā)現(xiàn)它可通過(guò)激活atm信號(hào)途徑來(lái)誘發(fā)dna損傷反應(yīng),并通過(guò)p53-p21通路引起細(xì)胞衰老。我們的這一發(fā)現(xiàn)具有以下兩點(diǎn)意義:首先,我們的研究表明,與促進(jìn)腫瘤細(xì)胞凋亡一樣,促進(jìn)腫瘤細(xì)胞的衰老同樣是一條值得探索和開(kāi)發(fā)的抗腫瘤途徑;其次,可以利用dznep能夠誘發(fā)dna損傷反應(yīng)這一特性,與放療、化療手段相結(jié)合,提高這些傳統(tǒng)的癌癥治療手段的效果。最后,本研究表明DZNep可以激活A(yù)TM,然而這種激活作用究竟是通過(guò)何種具體方式(DNA斷裂、ROS增加、染色質(zhì)狀態(tài)改變等),還有待于進(jìn)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.).
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.7
本文編號(hào)：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.).
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.7
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2 ;Overexpression of polo-like kinase1 predicts a poor prognosis in hepatocellular carcinoma patients[J];World Journal of Gastroenterology;2009年33期
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