【作者】 王阳；

【导师】 刘明耀； 逄秀凤；

【作者基本信息】 华东师范大学 ， 生物医学， 2014， 博士

【摘要】 Ⅰ.两种天然化合物通过下调CXCR4抑制肿瘤转移在癌症导致的死亡中,90%与肿瘤转移相关。最近,人们发现了一些与肿瘤靶向性迁移相关的分子机制。CXCR4是最初被发现于白细胞中并介导白细胞迁移的趋化因子受体,研究证实CXCR4通过与其专一性配体CXCL12作用还可介导肿瘤细胞向远端靶器官的迁移。目前CXCR4拮抗剂,如AMD3100和ALX40-4C,已进入临床用于癌症治疗。然而,这类拮抗剂只能靶向抑制定位于细胞膜上的功能性CXCR4受体,极易产生脱靶效应。因此,寻找或开发其他作用方式的CXCR4小分子抑制剂具有重要价值。本研究发现了两种天然化合物能抑制多种肿瘤细胞中CXCR4的表达,且抑制效应随用药浓度和药物作用时间增加而增强,这两种天然化合物分别为啤酒花雌性花序提取的异戊烯类黄酮化合物黄腐酚以及树胶脂中提取的乳香脂酸衍生物11-羰基-p-乙酰乳香酸。为探究两者降低CXCR4蛋白水平的机制,我们首先用蛋白酶体及溶酶体抑制剂提前处理细胞以阻断CXCR4降解途径。结果显示,这些抑制剂并不能回复黄腐酚及11-羰基-p-乙酰乳香酸对CXCR4的抑制作用,说明两者并非通过促进CXCR4降解而下调其表达,荧光定量PCR结果显示两者对CXCR4表达的抑制发生于转录水平。随后的电泳迁移率变动分析实验及染色质免疫共沉淀实验进一步揭示了黄腐酚和11-羰基-p-乙酰乳香酸均能有效下调CXCR4最主要的转录因子NF-κB的活性及NF-κB与CXCR4启动子区域的结合。通过降低CXCR4的表达,黄腐酚和11-羰基-p-乙酰乳香酸均能显著抑制由CXCL12介导的肿瘤细胞的迁移及趋化运动能力。黄腐酚还具有两种结构类似物。尽管三者共存于啤酒花中,而黄腐酚是三者中唯一能下调CXCR4表达的化合物。本研究提示,作为新型的CXCR4抑制剂,黄腐酚和11-羰基-p-乙酰乳香酸很有可能成为治疗癌症转移的潜在候选化合物。Ⅱ.放线菌素D通过靶向瓦博格效应抑制非小细胞肺癌生长肺癌是目前全球范围内致死率最高的癌症,其中多以非小细胞肺癌为主。有氧糖酵解即瓦博格效应作为包括非小细胞肺癌在内的大多数实体瘤的异常代谢方式,目前已成为肿瘤治疗的新靶点,而缺氧诱导因子1(HI-F1)是糖酵解通路的最主要的调控因子。由于目前处于临床试验阶段的糖酵解抑制剂存在诸多问题,因此亟需寻找高效、稳定、安全的新型抑制剂以靶向瓦博格效应治疗非小细胞肺癌。FDA批准的药物具有较好的安全性、详细的药物代谢动力学相关信息及完善的生产和销售网络等诸多优势,通过“药物重新利用”,能大大缩短药物研发周期。本研究旨在通过对FDA药库的筛选以期找到具有影响肿瘤细胞异常代谢的新功能或新机制的老药。本文首先用细胞存活率实验筛选了含1280个小分子化合物的FDA药库,排除了在50μM初筛浓度下细胞存活率仍大于60%的药物,而后将剩下的188个药物在低于IC50的浓度下通过瓦博格效应特征性表型之一的乳酸分泌实验进一步筛选,从中挑选得到4个备选药物并检测了它们对细胞糖酵解代谢相关的其他表型的影响,包括葡萄糖吸收、胞外酸化速率、细胞的糖酵解能力、ATP产出以及细胞氧消耗速率,其中,放线菌素D能浓度梯度依赖性地抑制细胞的乳酸分泌及上述糖酵解代谢相关表型。在随后的小鼠体内肿瘤模型及小动物PET/CT实验中,放线菌素D在60 gg/kg、一周两次、尾静脉注射的方式下给药30天,不但有效减缓小鼠体内肿瘤生长,控制肿瘤的重量、体积及大小,更显著抑制了肿瘤对葡萄糖的吸收能力。通过综合分析表型实验及动物模型实验的结果,本文选择放线菌素D进行下一步机制研究。放线菌素D是从土壤中链霉菌属的细菌分离出来的放线菌素类多肽类抗生素中最重要的一种,抑制RNA的合成是其原始机制。本研究发现放线菌素D在有无氯化钴存在的情况下均能浓度梯度及时间梯度依赖性地抑制HIF1因子下游糖酵解通路中多个关键激酶mRNA水平的表达,该抑制机制并不在于放线菌素D对HIF1因子蛋白表达水平的影响,而是抑制了HIF1因子的活性。核质分离实验结果表明,放线菌素D能有效抑制氯化钴刺激下HI1α的入核,而在不加氯化钴的情况下,该效果却并不明显。进一步的免疫共沉淀实验结果显示放线菌素D能显著地抑制HIF1α与丙酮酸激酶M2(PKM2)的结合,使得调控糖酵解通路相关基因的转录因子复合体不能形成,从而导致糖酵解通路中重要激酶的表达受阻。可见,放线菌素D抑制肿瘤的瓦博格效应主要是通过影响HIF1α在核内的定位及其与PKM2也的结合两方面机制共同实现的。本研究发现了放线菌素D抑制肿瘤异常代谢的新机制,为今后放线菌素D的临床应用提供了有价值的理论指导。更多还原

【Abstract】 Ⅰ. Two natural compounds Suppresses Cancer Cell Invasion through Inhibiting the Expression of CXCR4 Chemokine ReceptorCancer metastasis is the main cause of death (90%), and only recently we have gained some insight into the mechanisms by which metastatic cells arise from primary tumors and target to specific organs. Cysteine X Cysteine (CXC) chemokine receptor 4 (CXCR4), initially linked with leukocyte trafficking, is overexpressed in various tumors and mediates homing of tumor cells to distant sites expressing its cognate ligand CXCL12. Currently, CXCR4 antagonists, such as AMD3100 and ALX40-4C have entered clinical use in cancer treatment. However, due to several clinical problems including off-target effects of current antagonists, identification of other CXCR4 inhibitors has great potential to abrogate tumor metastasis.In this study, we demonstrated that xanthohumol (XN), a prenylflavonoid derived from the female flowers of the hops plant (Humulus lupulus. L), and acetyl-11-keto-β-boswellic acid (AKBA), a component in gum resin of therapeutic plant Boswellia serrata, suppressed CXCR4 expression in various cancer cell types in a concentration- and time-dependent manner. Both proteasome and lysosomal inhibitors had no effect to prevent the downregulation of CXCR4, suggesting that their inhibitory effect was not due to proteolytic degradation but occurred at the transcriptional level. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay further confirmed that they could block endogenous activation of nuclear factor kappa B, a key transcription factor regulating the expression of CXCR4 in cancer cells. Consistent with the above molecular basis, XN and AKBA remarkably abolished CXCL12-induced cancer cell invasion. Interestingly, although co-exist in hops, XN was the only compound that exhibited the inhibitory effect on the expression of CXCR4 compared with other analogues, isoxanthohumol and 8-prenylnaringenin.Together, our results suggeste that XN and AKBA, as novel inhibitors of CXCR4, could be a promising cancer therapeutic agent.Ⅱ. A FDA-approved drug-actinomycin D inhibit the growth of non-small cell lung cancer by targeting the warburg effectCurrently, Lung cancer is the leading cause of cancer mortality worldwide, and most of them (85%) are classified as non-small cell lung cancer. Warburg effect (aerobic glycolysis) as major abnormal metabolism existing in the majority of solid tumors, including non-small cell lung cancer, has become a new target for cancer therapy. Among tens of regulators in Warburg effect, HIF1 is the most important one in this process. However, due to several clinical problems of current glycolytic inhibitors, it is urgently needed to identify novel, effective and safe inhibitors targeting Warburg effect for non-small cell lung cancer treatment. FDA-approved drugs possess well-established safety and pharmacokinetic profiles, as well as manufacturing and distribution networks. Therefore, by screening these drugs, we can greatly shorten the period of novel drug discovery. The goal of this study is to identify old FDA-approved drugs with new functions targeting tumor abnormal metabolism.We first screened 1280 FDA-approved small molecules by cell viability assay and excluded those drugs with cancer cell inhibitory rates lower than 40% (at dose of 50 μM). One hundred and eighty eight drugs were left for lactate release analysis and 4 candidates were screened out. The effects of these 4 drugs on glucose uptake, extracellular acidification rate, glycolytic capacity of cells, ATP production and cellular oxygen consumption rate were further evaluated. Actinomycin D effectively suppressed tumor glycolytic phynotypes in vitro in a concentration-dependent manner. Additionally, at a dose of 60 μg/kg i.v. twice a week for 30 days, actinomycin D remarkably inhibited tumor growth and glucose uptake of the tumor tissue, indicating actinomycin D targeting warburg effect. Thus, we chose actinomycin D as drug candidate and for detailed mechanism study.Actinomycin D was one of the most important peptide antibiotics isolated from soil bacteria of the genus Streptomyces, and was originally shown to have the ability to inhibit transcriptional activity. In this study, we found that actinomycin D, with or without cobalt chloride, could inhibit the expression of the key kinases downstream of HIF1 in glycolytic pathway with a concentration- and time-dependent manner. The mechanism of inhibition did not occur at HIF1 protein levels, but was due to the downregulation of HIF1 transcription activity. Through nuclear/cytoplast separation, we confirmed that actinomycin D blocked HIF1 a translocation into the nucleus in the presence of cobalt chloride. Further co-immunoprecipitation study showed actinomycin D significantly attenuated the interaction of HIF1 a and PKM2, which hindered the formation of HIF1 transcriptional complex, resulting in the blocking of the expression of key kinases in glycolytic pathway.In this study, we found that actinomycin D inhibited tumor glycolysis by interfering with HIFla pathway. Our results provides valuable theoretical guidance for clinical applications of actinomycin D.更多还原