發(fā)布時間：2018-10-14 17:28

【摘要】：組蛋白去乙酰化酶(HDACs)是一類負(fù)責(zé)組蛋白表觀遺傳修飾的zn2+離子蛋白酶。HDACs表達(dá)或活化異常多與腫瘤的惡性程度及不良預(yù)后相關(guān)。組蛋白去乙�；敢种苿�(HDACIs)能夠抑制IDACs的活性,重啟癌癥相關(guān)基因表達(dá),因而能有效抑制腫瘤生長、誘導(dǎo)腫瘤細(xì)胞死亡。HDACIs陽性反應(yīng)率高、腫瘤細(xì)胞選擇性強(qiáng),是極具開發(fā)潛力的抗腫瘤制劑。本研究意在從備選化合物庫中篩選出腫瘤細(xì)胞增殖抑制活性高的HDACIs單體,并以此化合物為誘導(dǎo)劑,明確環(huán)肽類HDACIs抑制腫瘤細(xì)胞增殖與遷移的相關(guān)機(jī)制,分析各相關(guān)調(diào)控因子的潛在作用方式。 本研究選取了多種腫瘤細(xì)胞系,通過細(xì)胞毒性、蛋白檢測、顯微成像技術(shù)等方法,證實CTS203(cyclo(-L-Asu(NHOH)-Am3c6c-L-Phe-D-Pro-))是一種有應(yīng)用前景的高效HDACIs。細(xì)胞周期分布以及相關(guān)調(diào)控因子的表達(dá)等檢測進(jìn)一步證實凋亡與細(xì)胞周期阻滯是CTS203發(fā)揮增殖作用的主要方式,且具有明顯的時間依賴性：隨著CTS203作用時間的延長,MCF-7細(xì)胞內(nèi)cyclin D1表達(dá)持續(xù)下調(diào)、p21表達(dá)逐漸增加,共同促進(jìn)細(xì)胞周期的阻滯；凋亡效應(yīng)因子caspase-3與細(xì)胞色素c的含量出現(xiàn)成倍升高,表明凋亡進(jìn)程被激活；caspase-8、caspase-9等凋亡通路表征蛋白出現(xiàn)不同程度的活化,表明CTS203主要誘導(dǎo)線粒體通路的凋亡。 隨后針對CTS203時間依賴性增殖抑制能力的形成機(jī)制進(jìn)行了研究。時序性采集的免疫熒光顯微圖像以及蛋白表達(dá)變化顯示,在CTS203作用初期,自噬削弱了CTS203誘導(dǎo)的細(xì)胞毒性作用。對自噬體數(shù)目以及胞內(nèi)自噬相關(guān)蛋白量的檢測顯示,抑制自噬可進(jìn)一步促進(jìn)CTS203誘導(dǎo)的細(xì)胞毒性作用,降低化合物劑量、縮短效應(yīng)時間。與此同時,對凋亡相關(guān)因子的檢測顯示,自噬抑制通過誘導(dǎo)Beclin-1的剪切并同時促進(jìn)caspase-8、 caspase-9的活化,進(jìn)而放大凋亡信號、增強(qiáng)細(xì)胞毒性。 本研究還證實了CTS203對腫瘤細(xì)胞侵襲、遷移及集落形成能力的抑制作用。動力學(xué)分析、明膠酶譜實驗以及蛋白表達(dá)變化的分析結(jié)果顯示,CTS203具有與MMP-2形成酶-抑制劑復(fù)合物的潛力,并可通過上調(diào)RECK蛋白以及TIMP-1/MMP-2的比例抑制MMP-2的胞外活性；劃痕實驗與軟瓊脂實驗結(jié)果顯示,CTS203可有效抑制腫瘤細(xì)胞侵襲、減少新生集落數(shù)量。 綜上,本研究篩選出具有多方面應(yīng)用前景的異羥肟酸類環(huán)肽衍生物CTS203。本文中對CTS203增效機(jī)制的探討,暗示CTS203與其他種類的抗腫瘤制劑間存在潛在的協(xié)同效應(yīng)；應(yīng)用分子對接與實驗相結(jié)合暗示CTS203具有與MMP-2結(jié)合的潛力,為MMP-2特異性抑制劑的開發(fā)提供了理論依據(jù),為明確HDACIs抑制腫瘤細(xì)胞遷移的潛在機(jī)制指明了新的探索方向。
[Abstract]:Histone deacetylase (HDACs) is a kind of zn2 ion protease responsible for histone epigenetic modification. Abnormal expression or activation of HDACs is associated with malignancy and poor prognosis of tumors. Histone deacetylase inhibitor (HDACIs) can inhibit the activity of IDACs and restart the expression of cancer-related genes, which can effectively inhibit tumor growth and induce tumor cell death. It is a potential anti-tumor preparation. The aim of this study was to screen out HDACIs monomers with high proliferation inhibition activity from alternative compounds library, and use these compounds as inducers to clarify the mechanism of cyclopeptide HDACIs inhibiting proliferation and migration of tumor cells. To analyze the potential action of the relevant regulatory factors. In this study, we selected a variety of tumor cell lines, and proved that CTS203 (cyclo (- L-Asu (NHOH)-Am3c6c-L-Phe-D-Pro- is a promising high-efficiency HDACIs. by cytotoxicity, protein detection and microscopic imaging Cell cycle distribution and the expression of related regulatory factors further confirmed that apoptosis and cell cycle arrest are the main ways of CTS203 to play a role in proliferation. With the prolongation of CTS203, the expression of cyclin D1 in MCF-7 cells continued to decrease, p21 expression increased gradually, and the apoptosis effector caspase-3 and cytochrome c increased exponentially. The results indicated that the apoptosis process was activated, and that caspase-8,caspase-9 and other apoptotic pathways were characterized by different degree of activation of proteins, indicating that CTS203 mainly induces apoptosis of mitochondrial pathway. Then the mechanism of time-dependent proliferation inhibition in CTS203 was studied. Sequential immunofluorescence microscopic images and changes in protein expression showed that autophagy weakened the cytotoxicity induced by CTS203 at the initial stage of CTS203. The number of autophagy and the amount of intracellular autophagy related protein were measured. The inhibition of autophagy could further promote the cytotoxicity induced by CTS203, reduce the dose of compounds and shorten the effect time. At the same time, the detection of apoptosis-related factors showed that autophagy inhibition enhanced the cytotoxicity by inducing the shearing of Beclin-1 and simultaneously promoting the activation of caspase-8, caspase-9. This study also confirmed the inhibitory effect of CTS203 on tumor cell invasion, migration and colony formation. Kinetic analysis, gelatinase assay and protein expression analysis showed that CTS203 had the potential to form an enzyme inhibitor complex with MMP-2, and could inhibit the extracellular activity of MMP-2 by up-regulating the ratio of RECK protein and TIMP-1/MMP-2. The results of scratch test and soft Agar test showed that CTS203 could effectively inhibit the invasion of tumor cells and reduce the number of new colonies. In summary, the CTS203. derivatives of hydroxamic acid cyclopeptide with various application prospects were screened out in this study. The discussion of the synergistic mechanism of CTS203 suggests that there is a potential synergistic effect between CTS203 and other kinds of antitumor agents, and the application of molecular docking and experiments suggests that CTS203 has the potential to bind to MMP-2. It provides a theoretical basis for the development of MMP-2 specific inhibitors and provides a new exploration direction for clarifying the potential mechanism of HDACIs inhibiting tumor cell migration.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R737.9

【參考文獻(xiàn)】

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1 馬健;趙名;于曉Y，

本文編號：2271126