四种单体成分对前列腺癌和乳腺癌细胞的作用机制研究

发布时间：2018-02-24 22:03

本文关键词： 细梗香草皂苷B和C 化合物EM340 Chalocomoracin 凋亡自噬粘附和转移 LATS1激酶 YAP蛋白 Hippo信号通路线粒体自噬类凋亡　出处：《浙江大学》2016年博士论文　论文类型：学位论文

【摘要】：肿瘤是危害人类健康的难治性多发病,其危害性仅次于心血管疾病,是导致国内人口死亡的第二大原因。由于肿瘤多重耐药性日益严重,寻找全新结构和作用机理的抗肿瘤药物显得尤为重要。本文分别对天然源的细梗香草皂苷B(CapilliposideB,CPS-B)、细梗香草皂苷 C(CapilliposideC,CPS-C)和Chalocomoracin(CMR),以及实验室合成化合物EM340进行了深入地抗肿瘤作用机制研究。第一部分是关于天然植物单体化合物CPS-B和CPS-C抗前列腺癌的机制研究。CPS-B和CPS-C是新型齐墩果烷型三萜皂苷,自植物细梗香草(Lysimachia capillipes Hemsl,L.capillipes)中分离获得。前期的体内实验结果显示,细梗香草总皂苷(LysimachiaCapillipes,LC)抗肿瘤作用显著;但对于其所含单体活性成分CPS-B和CPS-C的抗肿瘤作用机制还不甚了解。本论文细胞存活率实验表明CPS-B和CPS-C对前列腺癌细胞PC-3和DU145的生长和增殖具有显著的抑制作用;经过48小时作用,CPS-B对PC-3和DU145的半数抑制浓度分别为3.75 μM和4.0μM,CPS-C对PC-3和DU145的半数抑制浓度分别为5.53μM和9.99μM。以PC-3细胞为研究对象,探究CPS-B和CPS-C抗前列腺癌作用机制,实验结果显示CPS-B通过引起细胞DNA损伤和凋亡,进而诱导细胞产生活性氧,激活AMPK-mTOR通路,引起细胞自噬以及细胞粘附和转移能力丧失最终导致细胞死亡;而CPS-C则通过作用于细胞线粒体,影响MAPK、Caspase和Bcl-2家族蛋白的表达而导致细胞凋亡性死亡。第二部分是关于化合物EM340抗乳腺癌的机制研究。本论文首先以YAP为靶目标,通过报告基因法筛选出有效抑制YAP表达的化合物EM340。进一步的实验结果显示,化合物EM340通过激活Hippo信号通路上游的LATS1激酶,引起Hippo通路中心信号蛋白YAP表达下调,诱导乳腺癌细胞的死亡。另一方面,通过阻断E3泛素化连接酶β-TRCP可有效抑制YAP的下降,说明EM340介导的Hippo信号通路的激活引起了 YAP的泛素化降解。采用RT-PCR和qPCR技术,发现YAP下游目标转录因子CTGF,Cyr61和干细胞相关因子Oct3/4,Sox2,Nanog的表达被抑制。总之,EM340以LATS1激酶为靶点作用于Hippo信号通路,进而引起Hippo下游效应子YAP降解,导致YAP下游目标转录因子表达下降,最终导致细胞死亡。第三部分是关于桑叶中Diels-Alder型化合物Chalocomoracin(CMR)抗前列腺癌和乳腺癌的机制研究。实验结果显示,CMR引起了 PC-3和MDA-MB-231细胞非凋亡性的细胞质空泡死亡现象(LNCaP细胞无此现象)。进一步的实验结果显示,CMR介导的PC-3和MDA-MB-231细胞活性氧增加、线粒体膜电位丧失以及钙离子失衡引起了细胞内质网应激,LC3-II、泛素化蛋白以及线粒体效应子PINK1的合成,导致细胞产生线粒体自噬;另一方面,CMR也引起了类凋亡抑制因子Alix表达的下调,提示CMR诱导细胞发生了类凋亡。对LNCaP在CMR作用下呈现的变化进行深入研究发现,PINK1在CMR介导的细胞自噬和类凋亡之间扮演着重要的角色。本论文主要的创新性工作如下:(1)首次较深入地研究了 CPS-B和CPS-C抗前列腺癌的作用机制。(2)基于YAP报告基因抗肿瘤化合物筛选,首次证明了EM340是LATS1激酶的激活剂。(3)CMR通过新颖的类凋亡途径介导了前列腺癌PC-3细胞和乳腺癌MDA-MB-231细胞的死亡。
[Abstract]:Tumor is a refractory disease endangering human health, its harm after cardiovascular disease, is the second leading cause of death. Because of the domestic population of multi drug resistance of tumor is becoming increasingly serious, looking for new structures and mechanisms of antitumor drugs is particularly important. This paper capilliposide B of natural sources (CapilliposideB CPS-B), capilliposide C (CapilliposideC, CPS-C) and Chalocomoracin (CMR), as well as the laboratory synthesis of EM340 were studied the antitumor mechanism deeply. The first part is about the research on the mechanism of.CPS-B and CPS-C natural plant compounds CPS-B and CPS-C against prostate cancer is the three new oleanane type saponins. Since the plant Lysimachia capillipes (Lysimachia capillipes, Hemsl, L.capillipes) isolated in vivo. Previous experimental results show that the total saponins of Lysimachia capillipes (LysimachiaCapil Lipes, LC) anti-tumor effect; but for the antitumor mechanism of the monomer containing active components CPS-B and CPS-C are not well understood. The experiment showed that the cell survival rate has significant inhibitory effect on CPS-B and CPS-C on growth and proliferation of cancer cells PC-3 and DU145 prostate; after 48 hours, CPS-B half of the PC-3 and DU145 inhibitory concentrations were 3.75 M and 4 M, CPS-C PC-3 and DU145 on the half inhibitory concentrations were 5.53 M and 9.99 M. in PC-3 cells as the research object, to explore CPS-B and CPS-C anti cancer mechanism of prostate. The experimental results show that the CPS-B caused by injury and apoptosis in DNA cells then, production of ROS induced by activation of the AMPK-mTOR pathway, induced, autophagy and cell adhesion and metastasis loss result in cell death; and CPS-C by acting on the cell mitochondria, effects of MAPK, Caspase and Bcl-2 The expression of proteins and induce apoptosis of death. The second part is about the research on the mechanism of compound EM340 against breast cancer. In this paper, YAP as a target, the reporter gene was screened effectively inhibit the expression of YAP compounds EM340. further experimental results show that the compound EM340 through activation of Hippo signaling pathway upstream of LATS1 kinase. Decrease the expression of Hippo pathway center signal protein YAP, induced cell death in breast cancer cells. On the other hand, the decline in the blocking of the E3 ubiquitin ligase beta -TRCP can effectively inhibit YAP activation, Hippo EM340 mediated signal pathway induced by the ubiquitination of YAP. Using RT-PCR and qPCR technology, YAP downstream target the transcription factor CTGF, Cyr61 and stem cell related factors Oct3/4, Sox2, Nanog expression was inhibited. In short, EM340 to LATS1 kinase targeting on Hippo signaling pathway in. Due to Hippo downstream effectors of YAP degradation, leading to YAP downstream targets of transcription factor expression decreased, eventually leading to cell death. The third part is about the Diels-Alder in mulberry leaves compound Chalocomoracin (CMR) mechanism of anti prostate cancer and breast cancer. The experimental results show that CMR caused PC-3 and MDA-MB-231 cell non apoptotic phenomenon of cytoplasmic vacuoles (LNCaP cell death without this phenomenon). The results showed that oxygen PC-3 and MDA-MB-231 cell activity of CMR mediated increase in mitochondrial membrane potential loss and calcium imbalance caused endoplasmic reticulum stress, LC3-II, ubiquitin protein synthesis and mitochondrial effector of PINK1, causing the cells to produce mitochondrial autophagy; on the other hand also, CMR caused a downregulation of apoptosis inhibitory factor Alix expression, suggesting that the occurrence of the apoptosis induced by CMR cells. The changes of LNCaP in the presence of CMR presentation Further study found that PINK1 plays an important role in autophagy and CMR mediated apoptosis. The main innovative work of this thesis are as follows: (1) the mechanism for the first time more in-depth study of the CPS-B and CPS-C against prostate cancer. (2) the YAP reporter gene based screening of antitumor compounds for the first time. It is proved that EM340 activator of LATS1 kinase. (3) CMR prostate cancer PC-3 cells and breast cancer MDA-MB-231 cell death by apoptosis mediated novel class.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.25;R737.9

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