吡唑环氧烷衍生物3g对结肠癌细胞自噬和细胞周期的调节及分子机制研究
发布时间：2018-08-02 21:43
【摘要】：研究背景和研究目的结肠癌在在人类常见的癌症中的被诊断率已经跃居第三位。在癌症的治疗中,除了局部切除治疗外,化疗仍然是一个普遍并有效的方式。许多联合给药的治疗方案中,化疗药物都具有一定的毒副作用。筛选新型小分子化合物抑制肿瘤细胞生长并且研究其具体机制仍然是研究中的热点。并且通过化学遗传学的方法研究细胞程序性死亡和细胞周期的具体机制能够为促进药物开发,为肿瘤治疗打下理论基础。细胞程序性死亡可以被分为三大类,凋亡是其中的一个重要类型,在多细胞生物的正常发育和稳态保持中有着关键的调控作用。凋亡是去除不需要的、衰老的和受损的细胞的重要机制,凋亡的失调会导致多种疾病,其中包括癌症的发展。自噬是另一类重要的细胞程序性死亡,在正常条件下可以通过降解多余的细胞器和细胞内错误折叠的蛋白等,来提供营养用以维持细胞正常的生命活动。但是在凋亡缺陷性细胞的死亡中,自噬又是细胞死亡必需的,这表明了自噬的双重性。自噬的调节是一个精密而复杂的基因调控过程,在各阶段都受到多种调节因子的作用。自噬可以分为mTOR依赖途径和mTOR非依赖途径,在前者中主要的分子机制是通过Akt/mTOR信号通路和其下游底物发挥作用,并参与调节翻译和细胞生长。而细胞自噬和周期也相互联系,并且mTOR在其中发挥重要作用。在我们实验室的前期工作中,发现在除血清和生长因子条件诱导的血管内皮细胞的凋亡过程中,小分子化合物3g通过诱导Ingerinβ4蛋白磷酸化并诱导入核,从而有效地诱导血管内皮细胞凋亡。但是其是否能够在肿瘤细胞中发挥抑制生长的作用从而成为一个有效的肿瘤治疗药物则还不清楚。而且其是否会对正常细胞的生长产生影响是寻找新型药物的重点之一,寻找到能够特异性抑制肿瘤细胞的化合物能够为药物开发提供新的前景。磷脂酰乙醇胺结合蛋白1(PEBP1),也被称为Raf激酶抑制蛋白,参与了MAPK,GPCR,NF-κB,GSK3β等多种细胞生长相关的信号通路的调控。PEBP1可以通过参与多种将细胞外刺激转化成不同信号以维持细胞完整性和体内平衡的通路。结肠癌患者的PEBP1启动子甲基化程度远远高于正常样本,这表明了其异常在结肠癌起始中潜在的重要性。而且最新的研究中PEBP1和LC3的直接相互作用得到了验证,PEBP1通过保守的LIR基序和LC3结合并且抑制营养剥夺的自噬,而且是通过PEBP1S153的磷酸化发挥作用的。本研究利用化学遗传学的原理与技术,以化合物小分子3g为研究工具,研究了其特异性抑制结肠癌细胞生长中的具体分子机制,确定了其在调控细胞自噬和细胞周期中发挥的作用,为结肠癌的治疗提供新的靶点和有效工具。研究方法1.人结肠癌Hct116细胞、人脐静脉血管内皮细胞、人肺腺癌A549细胞、人宫颈癌HeLa细胞、人前列腺癌PC3细胞的培养2.倒置相差显微镜观察细胞形态学变化3.SRB法检测细胞存活率4.Heochest染色后,通过荧光显微镜观察细胞核形态的变化,从而判断细胞是否凋亡5.LDH(乳酸脱氢酶)检测细胞是否发生坏死6.流式细胞术检测细胞凋亡和细胞周期7.激光共聚焦显微镜检测LC3-II的分布8.western blot检测PARP和caspase-3蛋白切割水平,LC3-II和p62蛋白表达水平,以及Akt,mTOR,P70S6K,4EBP1,PEBP1的磷酸化水平9.鸡胚尿囊膜人移植瘤模型研究小分子化合物对实体瘤进展和正常血管生成的影响研究结果1.吡唑环氧烷衍生物具有对多种肿瘤细胞的生长抑制作用1.1 SRB检测结果表明,小分子化合物3g(1-10 μM)作用于多种肿瘤细胞后,均能显著抑制细胞的存活。其中,化合物对结肠癌细胞Hct116细胞的抑制作用最强。并且在同样浓度下不影响正常培养条件的血管内皮细胞生长。1.2倒置相差显微镜观察细胞形态发现,化合物3g(2.5-10 μM)处理Hct1 16细胞24h后,细胞逐渐变圆。而3g处理A549细胞24h后,细胞发生了明显的拉长。1.3 Heochest染色结合荧光显微镜观察化合物3g(2.5-10 μM)处理肿瘤细胞24 h后,核凝集现象与对照组相比变化不明显。1.4流式细胞术结果表明,化合物3g(5μM)处理Hct116细胞24h后,处理组与对照组相比凋亡率没有发现显著升高1.5 Western-blot 检测在 12h、24h、48h 时 3g(5 μM)可能不能诱导 PARP 和caspase-3切割上调。1.6 LDH检测发现,化合物3g(10 μM)处理多种肿瘤细胞48 h后,培养液上清中LDH活性没有显著性差异,表明细胞没有发生坏死。2.小分子化合物3g通过调节细胞自噬和细胞周期发挥抑制作用2.1通过流式细胞术发现3g(5μM)处理Hct116细胞24h后会引起G1期细胞阻滞。2.2 western blot结果表明,设计不同的时间点使用3g(5 μM)处理Hct1 16细胞后,与对照组相比,3h、6h时Hct116细胞中LC3-Ⅱ表达显著增强,而在48h时自噬流被明显阻断。2.3免疫细胞化学检测发现,3g(5 μM)处理Hct116细胞3h后,与对照组相比,Hct116细胞中LC3出现了点状聚集。2.4 western blot结果表明,使用3g(5μM)处理Hct116细胞后,与对照组相比,短时间内Hct1 16细胞中Akt和mTOR的磷酸化被抑制。2.5 western blot结果表明,使用3g(5 μM)处理Hct1 16细胞后,与对照组相比,短时间内Hct116细胞中mTOR的底物P70S6K和4EBP1的磷酸化被抑制。2.6 Western blot结果表明,使用3g(5 μM)处理H116细胞后,与对照组相比,短时间内Hct1 16细胞中PEBP1的磷酸化被抑制。2.7鸡胚尿囊膜人移植瘤模型在3g处理后,对比对照组,处理组的实体瘤发展被明显抑制,并且不影响其正常血管的发生。结论1.小分子化合物3g能够显著抑制多种人肿瘤细胞存活,且抑制作用呈现浓度的依赖性,其中对Hct116细胞的抑制作用最强,并且在同样浓度下不影响正常培养液培养的人脐静脉血管内皮细胞。2.小分子化合物3g不是通过诱导凋亡来抑制Hct1 16细胞的生长。小分子化合物3g能够在短时期内诱导自噬,而在长时间时阻断自噬流,并且发挥诱导细胞周期阻滞的作用。其具体分子机制是通过在短期内诱导PEBP1 S153位点的磷酸化,并抑制Akt/mTOR信号通路实现的。PEBP1的磷酸化可能能够参与调解mTOR依赖的细胞自噬。3.小分子化合物3g能够在鸡胚尿囊膜人移植瘤模型中抑制实体瘤的发展,说明其有发展成为肿瘤抑制药物的前景。
[Abstract]:Background and research objectives of colon cancer are third in the diagnosis of human cancer. In the treatment of cancer, chemotherapy is still a common and effective way in addition to local resection. Subcompounds inhibit the growth of tumor cells and study their specific mechanism is still a hot spot in the study. And the specific mechanism of cell programmed cell death and cell cycle can be studied by chemical genetics to promote drug development and to lay a theoretical foundation for cancer treatment. Cell programmed death can be divided into three major categories, apoptosis It is one of the important types in which there is a key regulatory role in the normal development and homeostasis of multicellular organisms. Apoptosis is an important mechanism for removing the non needed, aging and damaged cells. The dysregulation of apoptosis can lead to a variety of diseases, including the development of cancer. Autophagy is another important type of cell programmed death. Normal conditions can be used to provide nutritional use to maintain normal cell life activities by degrading superfluous organelles and incorrectly folding proteins in cells. However, autophagy is essential for cell death in the death of apoptotic cells, which indicates the dual nature of autophagy. Autophagy is a precise and complex gene. The regulation process is affected by a variety of regulatory factors at all stages. Autophagy can be divided into mTOR dependent and mTOR non dependent pathways. In the former, the main molecular mechanism is to play the role of the Akt/mTOR signaling pathway and its downstream substrate, and to regulate translation and cell growth. In our previous work, we found that during the apoptosis of vascular endothelial cells induced by sera and growth factors, the small molecule compound 3G could induce Ingerin beta 4 protein phosphorylation and inducement, which could effectively induce the apoptosis of vascular endothelial cells. It is not clear that cells can play a role in inhibiting growth and become an effective tumor therapy drug. And whether it will affect the growth of normal cells is one of the key points for finding new drugs, and finding compounds that can specifically inhibit tumor cells can provide new prospects for drug development. Amine binding protein 1 (PEBP1), also known as Raf kinase suppressor, participates in the regulation of a variety of cell growth related signaling pathways such as MAPK, GPCR, NF- kappa B, GSK3 beta and other signaling pathways..PEBP1 can be involved in a variety of pathways that convert extracellular stimuli into different signals to maintain cell integrity and body balance. PEBP1 start Zi Jiaji in colon cancer patients This shows the potential importance of the abnormalities in the initiation of colon cancer. And in the latest studies, the direct interaction between PEBP1 and LC3 is verified, and PEBP1 combines the conservative LIR motif with LC3 and inhibits the autophagy of nutritional deprivation, and is mediated by the phosphorylation of PEBP1S153. The principles and techniques of chemical genetics are used to study the specific molecular mechanism of the small molecule 3G, which inhibits the growth of colon cancer cells, and determine its role in the regulation of autophagy and cell cycle, and provide new targets and effective tools for the treatment of colon cancer. 1. research methods have been made. Colon cancer Hct116 cells, human umbilical vein endothelial cells, human lung adenocarcinoma A549 cells, human cervical cancer HeLa cells, human prostate cancer PC3 cells, 2. inverted phase contrast microscope observation cell morphological changes, 3.SRB method to detect cell survival rate 4.Heochest staining, by fluorescence microscopy to observe the change of cell nuclear morphology, so as to judge the fine. Whether cell apoptosis 5.LDH (lactate dehydrogenase) detected cell necrosis and 6. flow cytometry to detect cell apoptosis and cell cycle 7. laser confocal microscopy detection of LC3-II distribution by 8.western blot detection of PARP and caspase-3 protein cutting levels, LC3-II and p62 protein expression levels, and Akt, mTOR, P70S6K, 4EBP1, phosphorylated water Study on the effect of small molecular compound on the progression of solid tumor and the formation of normal angiogenesis in the human fetal allantoic membrane model of 9. chick embryo. 1. the inhibitory effect of pyrazoloxane derivatives on the growth of various tumor cells was 1.1 SRB. The results showed that the small molecule compound 3G (1-10 mu M) could be significantly inhibited after a variety of tumor cells. The inhibitory effect of the compound on the colon cancer cell Hct116 cells was the strongest, and the cell morphology of the vascular endothelial cells, which did not affect the normal culture conditions under the same concentration, was observed by the.1.2 inversion phase contrast microscope, and the compound 3G (2.5-10 mu M) treated Hct1 16 cells 24h, and the cells gradually turned round. 3G treated A549. After the cell 24h, the cells had an obvious elongated.1.3 Heochest staining combined with the fluorescence microscope to observe the compound 3G (2.5-10 micron M) treatment of the tumor cell 24 h, and the nuclear agglutination was not obvious compared with the control group. The result of.1.4 flow cytometry showed that the compound 3G (5 micron M) treated Hct116 cell 24h, compared with the control group, the rate of apoptosis was no more than that of the control group. There was a significant increase of 1.5 Western-blot detection at 12h, 24h, and 48h 3G (5 M) may not induce PARP and caspase-3 cutting up up.1.6 LDH detection, compound 3G (10 micron) treatment of a variety of tumor cells 48, there is no significant difference in the activity of the culture liquid supernatant. Overregulation of autophagy and cell cycle inhibits the cell cycle 2.1 through flow cytometry that 3G (5 M) treatment of Hct116 cells 24h will cause G1 phase cell block.2.2 Western blot results, and the design of different time points using 3G (5 micron M) treatment Hct1 16 cells, compared with the control group, the expression significantly increased At the time of 48h, autophagic flow was obviously blocked by.2.3 immunocytochemical detection, and 3G (5 mu M) treated Hct116 cells 3H. Compared with the control group, LC3 appeared in Hct116 cells, and.2.4 Western blot results showed that after treating the cells with 3G (5 mu), compared with the control group, the 16 cells in a short period of time were phosphoric acid and phosphoric acid. The inhibition of.2.5 Western blot results showed that after Hct1 16 cells were treated with 3G (5 mu M), the phosphorylation of mTOR substrate P70S6K and 4EBP1 in Hct116 cells in a short time compared with the control group was suppressed.2.6 Western results showed that the phosphorous of 16 cells in a short time compared with the control group was compared with the control group. .2.7 chick embryo allantoic membrane human transplanted tumor model was suppressed after 3G treatment, compared with the control group, the development of solid tumor in the treatment group was obviously inhibited, and it did not affect the occurrence of normal blood vessels. Conclusion 1. small molecule compound 3G can significantly inhibit the survival of various human tumor cells, and the inhibitory production concentration dependence, among them, Hct116 thin The inhibitory effect of the cell is the strongest, and the.2. small molecule compound, 3G, which does not affect the normal culture medium, does not inhibit the growth of Hct1 16 cells by inducing apoptosis. The small molecule compound 3G can induce autophagy in a short period of time, and the autophagy is blocked at a long time, and the induction of autophagy can be induced. The role of cell cycle arrest. Its specific molecular mechanism is the phosphorylation of the PEBP1 S153 site in the short term, and the phosphorylation of the.PEBP1, realized by the Akt/mTOR signaling pathway, may be able to participate in the mediation of the mTOR dependent cell autophagic.3. small molecule compound 3G to inhibit the growth of solid tumor in the human embryo transfer tumor model of the chicken embryo. It shows that it has the prospect of developing tumor suppressor drugs.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35
[Abstract]:Background and research objectives of colon cancer are third in the diagnosis of human cancer. In the treatment of cancer, chemotherapy is still a common and effective way in addition to local resection. Subcompounds inhibit the growth of tumor cells and study their specific mechanism is still a hot spot in the study. And the specific mechanism of cell programmed cell death and cell cycle can be studied by chemical genetics to promote drug development and to lay a theoretical foundation for cancer treatment. Cell programmed death can be divided into three major categories, apoptosis It is one of the important types in which there is a key regulatory role in the normal development and homeostasis of multicellular organisms. Apoptosis is an important mechanism for removing the non needed, aging and damaged cells. The dysregulation of apoptosis can lead to a variety of diseases, including the development of cancer. Autophagy is another important type of cell programmed death. Normal conditions can be used to provide nutritional use to maintain normal cell life activities by degrading superfluous organelles and incorrectly folding proteins in cells. However, autophagy is essential for cell death in the death of apoptotic cells, which indicates the dual nature of autophagy. Autophagy is a precise and complex gene. The regulation process is affected by a variety of regulatory factors at all stages. Autophagy can be divided into mTOR dependent and mTOR non dependent pathways. In the former, the main molecular mechanism is to play the role of the Akt/mTOR signaling pathway and its downstream substrate, and to regulate translation and cell growth. In our previous work, we found that during the apoptosis of vascular endothelial cells induced by sera and growth factors, the small molecule compound 3G could induce Ingerin beta 4 protein phosphorylation and inducement, which could effectively induce the apoptosis of vascular endothelial cells. It is not clear that cells can play a role in inhibiting growth and become an effective tumor therapy drug. And whether it will affect the growth of normal cells is one of the key points for finding new drugs, and finding compounds that can specifically inhibit tumor cells can provide new prospects for drug development. Amine binding protein 1 (PEBP1), also known as Raf kinase suppressor, participates in the regulation of a variety of cell growth related signaling pathways such as MAPK, GPCR, NF- kappa B, GSK3 beta and other signaling pathways..PEBP1 can be involved in a variety of pathways that convert extracellular stimuli into different signals to maintain cell integrity and body balance. PEBP1 start Zi Jiaji in colon cancer patients This shows the potential importance of the abnormalities in the initiation of colon cancer. And in the latest studies, the direct interaction between PEBP1 and LC3 is verified, and PEBP1 combines the conservative LIR motif with LC3 and inhibits the autophagy of nutritional deprivation, and is mediated by the phosphorylation of PEBP1S153. The principles and techniques of chemical genetics are used to study the specific molecular mechanism of the small molecule 3G, which inhibits the growth of colon cancer cells, and determine its role in the regulation of autophagy and cell cycle, and provide new targets and effective tools for the treatment of colon cancer. 1. research methods have been made. Colon cancer Hct116 cells, human umbilical vein endothelial cells, human lung adenocarcinoma A549 cells, human cervical cancer HeLa cells, human prostate cancer PC3 cells, 2. inverted phase contrast microscope observation cell morphological changes, 3.SRB method to detect cell survival rate 4.Heochest staining, by fluorescence microscopy to observe the change of cell nuclear morphology, so as to judge the fine. Whether cell apoptosis 5.LDH (lactate dehydrogenase) detected cell necrosis and 6. flow cytometry to detect cell apoptosis and cell cycle 7. laser confocal microscopy detection of LC3-II distribution by 8.western blot detection of PARP and caspase-3 protein cutting levels, LC3-II and p62 protein expression levels, and Akt, mTOR, P70S6K, 4EBP1, phosphorylated water Study on the effect of small molecular compound on the progression of solid tumor and the formation of normal angiogenesis in the human fetal allantoic membrane model of 9. chick embryo. 1. the inhibitory effect of pyrazoloxane derivatives on the growth of various tumor cells was 1.1 SRB. The results showed that the small molecule compound 3G (1-10 mu M) could be significantly inhibited after a variety of tumor cells. The inhibitory effect of the compound on the colon cancer cell Hct116 cells was the strongest, and the cell morphology of the vascular endothelial cells, which did not affect the normal culture conditions under the same concentration, was observed by the.1.2 inversion phase contrast microscope, and the compound 3G (2.5-10 mu M) treated Hct1 16 cells 24h, and the cells gradually turned round. 3G treated A549. After the cell 24h, the cells had an obvious elongated.1.3 Heochest staining combined with the fluorescence microscope to observe the compound 3G (2.5-10 micron M) treatment of the tumor cell 24 h, and the nuclear agglutination was not obvious compared with the control group. The result of.1.4 flow cytometry showed that the compound 3G (5 micron M) treated Hct116 cell 24h, compared with the control group, the rate of apoptosis was no more than that of the control group. There was a significant increase of 1.5 Western-blot detection at 12h, 24h, and 48h 3G (5 M) may not induce PARP and caspase-3 cutting up up.1.6 LDH detection, compound 3G (10 micron) treatment of a variety of tumor cells 48, there is no significant difference in the activity of the culture liquid supernatant. Overregulation of autophagy and cell cycle inhibits the cell cycle 2.1 through flow cytometry that 3G (5 M) treatment of Hct116 cells 24h will cause G1 phase cell block.2.2 Western blot results, and the design of different time points using 3G (5 micron M) treatment Hct1 16 cells, compared with the control group, the expression significantly increased At the time of 48h, autophagic flow was obviously blocked by.2.3 immunocytochemical detection, and 3G (5 mu M) treated Hct116 cells 3H. Compared with the control group, LC3 appeared in Hct116 cells, and.2.4 Western blot results showed that after treating the cells with 3G (5 mu), compared with the control group, the 16 cells in a short period of time were phosphoric acid and phosphoric acid. The inhibition of.2.5 Western blot results showed that after Hct1 16 cells were treated with 3G (5 mu M), the phosphorylation of mTOR substrate P70S6K and 4EBP1 in Hct116 cells in a short time compared with the control group was suppressed.2.6 Western results showed that the phosphorous of 16 cells in a short time compared with the control group was compared with the control group. .2.7 chick embryo allantoic membrane human transplanted tumor model was suppressed after 3G treatment, compared with the control group, the development of solid tumor in the treatment group was obviously inhibited, and it did not affect the occurrence of normal blood vessels. Conclusion 1. small molecule compound 3G can significantly inhibit the survival of various human tumor cells, and the inhibitory production concentration dependence, among them, Hct116 thin The inhibitory effect of the cell is the strongest, and the.2. small molecule compound, 3G, which does not affect the normal culture medium, does not inhibit the growth of Hct1 16 cells by inducing apoptosis. The small molecule compound 3G can induce autophagy in a short period of time, and the autophagy is blocked at a long time, and the induction of autophagy can be induced. The role of cell cycle arrest. Its specific molecular mechanism is the phosphorylation of the PEBP1 S153 site in the short term, and the phosphorylation of the.PEBP1, realized by the Akt/mTOR signaling pathway, may be able to participate in the mediation of the mTOR dependent cell autophagic.3. small molecule compound 3G to inhibit the growth of solid tumor in the human embryo transfer tumor model of the chicken embryo. It shows that it has the prospect of developing tumor suppressor drugs.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.35
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6 杨键;邓玉杰;张晓燕;吕鹏飞;徐俊;杨颖;宁光;;脂肪细胞自噬检测方法的建立及意义探讨[A];中华医学会糖尿病学分会第十六次全国学术会议论文集[C];2012年
7 赵颖;杨静;廖文娟;刘向宇;张辉;王杉;王冬来;冯京南;俞立;朱卫国;;胞浆中Fox01引起细胞自噬进而发挥抑制肿瘤的功能[A];中华医学会肿瘤学分会第七届全国中青年肿瘤学术会议——中华医学会肿瘤学分会“中华肿瘤 明日之星”大型评选活动暨中青年委员全国遴选论文汇编[C];2011年
8 陈永;邹s舠，
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