新型环肽类组蛋白去乙酰化酶抑制剂对乳腺癌细胞增殖与迁移的影响
发布时间：2018-10-14 17:28
【摘要】：组蛋白去乙酰化酶(HDACs)是一类负责组蛋白表观遗传修饰的zn2+离子蛋白酶。HDACs表达或活化异常多与肿瘤的恶性程度及不良预后相关。组蛋白去乙酰化酶抑制剂(HDACIs)能够抑制IDACs的活性,重启癌症相关基因表达,因而能有效抑制肿瘤生长、诱导肿瘤细胞死亡。HDACIs阳性反应率高、肿瘤细胞选择性强,是极具开发潜力的抗肿瘤制剂。本研究意在从备选化合物库中筛选出肿瘤细胞增殖抑制活性高的HDACIs单体,并以此化合物为诱导剂,明确环肽类HDACIs抑制肿瘤细胞增殖与迁移的相关机制,分析各相关调控因子的潜在作用方式。 本研究选取了多种肿瘤细胞系,通过细胞毒性、蛋白检测、显微成像技术等方法,证实CTS203(cyclo(-L-Asu(NHOH)-Am3c6c-L-Phe-D-Pro-))是一种有应用前景的高效HDACIs。细胞周期分布以及相关调控因子的表达等检测进一步证实凋亡与细胞周期阻滞是CTS203发挥增殖作用的主要方式,且具有明显的时间依赖性：随着CTS203作用时间的延长,MCF-7细胞内cyclin D1表达持续下调、p21表达逐渐增加,共同促进细胞周期的阻滞；凋亡效应因子caspase-3与细胞色素c的含量出现成倍升高,表明凋亡进程被激活；caspase-8、caspase-9等凋亡通路表征蛋白出现不同程度的活化,表明CTS203主要诱导线粒体通路的凋亡。 随后针对CTS203时间依赖性增殖抑制能力的形成机制进行了研究。时序性采集的免疫荧光显微图像以及蛋白表达变化显示,在CTS203作用初期,自噬削弱了CTS203诱导的细胞毒性作用。对自噬体数目以及胞内自噬相关蛋白量的检测显示,抑制自噬可进一步促进CTS203诱导的细胞毒性作用,降低化合物剂量、缩短效应时间。与此同时,对凋亡相关因子的检测显示,自噬抑制通过诱导Beclin-1的剪切并同时促进caspase-8、 caspase-9的活化,进而放大凋亡信号、增强细胞毒性。 本研究还证实了CTS203对肿瘤细胞侵袭、迁移及集落形成能力的抑制作用。动力学分析、明胶酶谱实验以及蛋白表达变化的分析结果显示,CTS203具有与MMP-2形成酶-抑制剂复合物的潜力,并可通过上调RECK蛋白以及TIMP-1/MMP-2的比例抑制MMP-2的胞外活性；划痕实验与软琼脂实验结果显示,CTS203可有效抑制肿瘤细胞侵袭、减少新生集落数量。 综上,本研究筛选出具有多方面应用前景的异羟肟酸类环肽衍生物CTS203。本文中对CTS203增效机制的探讨,暗示CTS203与其他种类的抗肿瘤制剂间存在潜在的协同效应；应用分子对接与实验相结合暗示CTS203具有与MMP-2结合的潜力,为MMP-2特异性抑制剂的开发提供了理论依据,为明确HDACIs抑制肿瘤细胞迁移的潜在机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.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9
[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.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R737.9
【参考文献】
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1 马健;赵名;于晓Y，
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