新型一氧化氮供体-β半乳糖基化偶氮烯翁二醇的抗菌、抗肿瘤作用途径研究

【作者】 陈畅；

【导师】 王鹏；

【作者基本信息】 山东大学 ， 微生物学， 2007， 博士

【摘要】 一氧化氮（NO）是一种在胞内由三种不同的一氧化氮合酶（iNOS、nNOS、eNOS）产生的气态小分子，它在松弛血管平滑肌、抑制血小板凝集、协助免疫系统破坏肿瘤细胞和胞内病原体（病毒和细菌）、参与神经突触传递等多种生理和病理的过程中起到了重要作用。一氧化氮具有极短的半衰期并且容易变成多种活性氮基团（RNS），如三氧化二氮（N₂O₃）和过氧化亚硝酸根离子（ONOO^-）等，因此在治疗和研究中利用这个活泼的小分子是很困难的。由于气态一氧化氮不稳定且不方便利用，人们对一氧化氮供体能在体内通过分解、氧化、还原的多种机制产生RNS产生了越来越浓的兴趣。NO供体是指能够储存并稳定释放活性氮基团（RNS）的一类有机化合物。以前应用常规NO供体作为药物前体遇到许多困难，例如药品的运输缺乏靶向性，难以达到胞内有效剂量以及有毒副作用等。迄今为止，许多NO供体的前体药物已经得到了发展，例如，亚硝基铁氰化钠（SNP），羟基脲以及合成的羟基脲衍生物，NONOates。但是至今大多数传统的NO供体的应用受到限制，主要因为缺乏特异性、缺乏适当的靶向传递途径，导致了许多副作用发生。因此，现在迫切需要设计出一种新型的能使NO靶向定点释放的NO供体，该供体能直接将可调节剂量的药物传递至位点作用。我们小组合成并报导的β-半乳糖基化偶氮烯翁二醇（β-Gal-NONOate）是一种新型定点释放NO的化合物，它一旦受到β-半乳糖苷酶的激活作用就能够释放RNS（NO）。1．在本研究中，我们将β-Gal=NONOate作为杀菌剂来研究其杀菌效果、作用机理。通过测定胞内的NO水平及比较用β-Gal-NONOate和NONOate分别处理的E．coli DH5α（pUC18）的存活率，我们可以很明显地观察到β-Gal-NONOate比传统的NONOate具有更高的杀菌活性。同时我们也比较了分别用β-Gal-NONOate和NONOate处理的E．coli DH5α的存活率，这二者都显示出较低的杀菌活性。这结果说明了β-Gal-NONOate是一个高效的并且很有前景的NO供体。β-Gal-NONOate被证实可借助于己糖载体很容易传送进入细菌胞内。因此，一旦内部细胞产生β-半乳糖苷酶就会引起β-Gal-NONOate的水解，NO也就能迅速地从该化合物上释放下来，在胞内达到有效浓度。证明了β-Gal-NONOate的释放方式是一种新颖、有效的在细菌胞内定点释放RNS（NO）的途径。2．基于以上研究，我们确认β-Gal-NONOate是一个有显著杀菌活性的定位胞内释放的NO供体，在临床应用和相关研究中都具有重要意义。第三章初步探索了β-Gal-NONOate在消除细菌普遍存在的抗生素耐药性方面的作用。首先，采用E-test法测定最低抑菌浓度（MIC）来确定原始菌株对抗生素的耐受度，经抗生素多代诱导后得到Amp耐药性菌株，再用β-Gal-NONOate与NONOate分别处理，在一系列连续的传代培养后使细菌恢复对抗生素的敏感性。研究结果显示，β-Gal-NONOate较之于NONOate，能更有效地降低Amp抗性菌株的MIC值，即消除了细菌的耐药性；当与Amp一起进行亚培养时，β-Gal-NONOate与NONOate相比，能在更短的代数内杀死细菌，也就是说大大的缩短了抗生素耐药性的持续时间。这些结果进一步证实了β-Gal-NONOate作为一种NO供体，不仅可被用作单纯的杀菌剂，还可用于消除目前日益严重的细菌的抗生素耐药性。基于前期的研究，我们断定β-Gal-NONOate通过向胞内运送更多的NO，依靠迅速达到有效浓度的NO消除了细菌对抗生素的耐药性。3．深入研究抗生素耐药性的分子机制有利于提高现有抗菌药物的作用效果。双向电泳（two dimensional electrophoresis，2-DE）是蛋白质组学研究的关键核心技术之一。第四章应用蛋白质组学方法比较分析了E．coli K-12中与Amp抗性产生和消除相关的蛋白质，以了解抗性产生的途径。利用2-DE技术比较了三种大肠杆菌菌株（E．coli K-12、E．coli K-12 Amp抗性菌、E．coli K-12 Amp抗性消除菌）相互间在蛋白质水平上的差异。分别提取三种菌的蛋白质，依次进行等电聚焦电泳（IFE）和十二烷基磺酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE），使用考马斯亮兰进行染色，经PDQuest7.2软件对结果进行比对、解析，以此研究β-Gal-NONOate对菌株在蛋白质水平上的影响。最终确认了β-Gal-NONOate可以通过改变细菌中蛋白质的水平来发挥消除Amp耐药性的作用。4．迄今为止，NO供体已经被用于抗肿瘤治疗的各个方面。为了能选择性地杀伤肿瘤细胞、避免不必要的副作用，第五章中β-Gal-NONOate被用到了抗肿瘤研究上。首次在9L／LacZ细胞中通过测定胞内释放NO的水平和抗肿瘤活性，证明了它更优于其前体NONOate。β-Gal-NONOate只在β-半乳糖苷酶激活下才会在9L／LacZ细胞内水解释放NO，这决定了它比NONOate具有更强的细胞毒性。结果显示：在9L／LacZ细胞内等浓度的情况下，β-Gal-NONOate产生的NO水平要高于NONOate，从而导致更高的抗肿瘤活性。然而，β-Gal-NONOate对9L细胞的毒性却不及NONOate。因此，β-Gal-NONOate是一种具有靶点特异性的药物前体，它是依赖于β-半乳糖苷酶的存在而发挥作用的定位胞内释放NO的NO供体，并很有希望成为一种有前途的探针和有潜力的新型抗肿瘤治疗药物。本研究首次设计并证明了一个新颖的肿瘤细胞内定点释放NO的药物传递途径，更容易使药物在胞内达到有效浓度，减毒增效降低毒副作用。这为新型抗肿瘤药物的开发提供了新的思路。5．第六章进一步比较研究了偶氮烯翁二醇（diazeniumdiolate，NONOate）与β-Gal-NONOate（β-galactosyl-diazeniumdiolate）的抗肿瘤作用。利用阳离子脂质体介导法，将真核表达载体pcDNA3-LacZ转染到HeLa细胞中；经G418（Geneticin）筛选，获得了稳定表达β-半乳糖苷酶的HeLa／LacZ细胞株。本研究由大鼠胶质瘤C6和C6／LacZ细胞，拓展到了人宫颈癌细胞模型，更全面地证实了β-Gal-NONOate的抗肿瘤作用和给药途径。本章通过Griess法比较了β-Gal-NONOate与NONOate在四种细胞中NO的释放量，根据细胞克隆形成率、细胞计数和MTT法比较β-Gal-NONOate与NONOate对细胞的抑制作用。结果显示β-Gal-NONOate在含有LacZ基因的两个细胞系中的NO释放量与药物浓度呈剂量依赖关系，并在相同药物浓度条件下明显高于NONOate；而β-Gal-NONOate在不表达β-半乳糖苷酶的C6、HeLa细胞中未检测到NO释放；β-Gal-NONOate对带有LacZ的两个细胞系的抗肿瘤活性显著高于NONOate（P＜0.05），但对C6、HeLa细胞则没有明显的抑制作用；NONOate在含或不含LacZ的四个细胞系中的NO释放量和抑制作用效果都没有显著差异。本章通过C6细胞及在人宫颈癌HeLa细胞中转入常见报告基因LacZ，再次证实了β-Gal-NONOate比NONOate更加稳定并具有更高的抗肿瘤活性，其作用的发挥依赖于对β-半乳糖苷酶的选择性，是一个有效的定位胞内释放的NO供体。作为抗肿瘤研究中的一种新型探针，本研究将β-Gal-NONOate的应用范围扩大到了对人肿瘤细胞的基因治疗层面上，预示了其未来在肿瘤的治疗和研究中的应用前景。6．第七章研究了新型NO供体β-Gal-NONOate对大鼠胶质瘤9L／LacZ、C6／LacZ细胞以及人子宫颈癌HeLa／LacZ细胞的诱导凋亡作用。将稳定表达β-半乳糖苷酶的三个细胞株经β-Gal-NONOate处理后，通过：光镜、荧光显微观察细胞形态学结构的改变和典型凋亡特征的出现、琼脂糖凝胶电泳观察凋亡典型的DNA ladder、原位末端标记法（TUNEL）检测凋亡细胞的DNA断裂、流式细胞仪（FCM）Annexin V-FITC／PI双染色测定细胞凋亡比例，证明了β-Gal-NONOate能够诱导三种细胞凋亡，药物处理组的细胞呈现出典型的凋亡特征，并且随着药物浓度和时间的增加，这种变化更加明显；细胞凋亡大于坏死，比例在一定范围内与药物浓度成正相关。应用激光共聚焦显微镜（LSCM）和特异荧光探针，对β-Gal-NONOate作用前后三种细胞内游离Ca²⁺浓度([Ca²⁺]_i)和线粒体跨膜电位（ΔΨm）的变化进行了精确测定。β-Gal-NONOate处理后，细胞内游离钙离子的荧光强度持续、较大幅度的升高，而线粒体跨膜电位迅速降低。由此可见，β-Gal-NONOate在胞内释放了NO后可能通过直接调节细胞膜上的Ca²⁺通道或同时在胞内释放内质网、线粒体等细胞内钙库中与钙结合蛋白松弛结合的游离钙，引起胞内[Ca²⁺]_i的变化，并将这种信号传递给线粒体。线粒体随即发生通透性变化，跨膜电位下降，引发线粒体功能改变，激活凋亡的关键酶Caspase级联放大，导致线粒体释放Cytc、ROS等一系列诱导凋亡因子。最后在Caspase、Bcl-2及核酸内切酶等的共同作用下，使细胞进入死亡程序。β-Gal-NONOate释放的NO或RNS可能进入细胞核，影响复制、转录、翻译等各个环节，进一步引起染色体的聚集、断裂、DNA ladder等凋亡特征性的形态和生化改变。本研究还利用MTT法证实了β-Gal-NONOate与常用抗肿瘤药cisplatin联用有增效作用（q＞1），这对临床抗肿瘤药物的减毒增效研究有重要的指导意义。更多还原

【Abstract】 NO, which is a paradoxical gaseous messenger enzymatically generated in vivo by three isoforms of nitric oxide synthase （iNOS, nNOS and eNOS）, plays an important role in numerous physiological and pathophysiological processes including relaxing vascular smooth muscle, inhibiting platelet aggregation, assisting the immune system in destroying tumor cells and intracellular pathogens and participating in neuronal synaptic transmission. Unfortunately, NO has extremely short half-life and is easily converted into a variety of reactive nitrogen species （RNS）, such as dinitrogen trioxide （N₂O₃）, nitrogen dioxide （NO₂）, and the peroxynitrite anion （ONOO^-）, so it is difficult to utilize this active small molecule in therapy and research. Thus site-specific delivery of exogenous NO becomes an attractive therapeutic option in the treatment of disease. Due to the instability and inconvenient handling of aqueous NO solutions, there has been an increasing interest in using NO donors capable of generating RNS in vivo by a variety of mechanisms including decomposition,oxidation, or reduction.1. β-galactosyl-pyrrolidinyl diazeniumdiolates （β-Gal-NONOate） is a newsite-specific NO-releasing compound reported by our group, which releases RNS（NO） once activated by β-galactosidase. In Chapter 2, β-Gal-NONOate was used as a bactericidal reagent to determine its effectiveness of NO releasing. Through the evaluation of intracellular NO level and the comparison of survival of E. coli transformed with lacZ gene but treated with β-Gal-NONOate and NONOate, respectively, it’s evident that β-Gal-NONOate had a higher bactericidal activity than conventional NONOate. While either β-Gal-NONOate- or NONOate-treated-E. coli without transferred lacZ gene manifested low bactericidal activity. The results revealed that β-Gal-NONOate was a high effective and promising NO donor. It was presumed that β-Gal-NONOate could be easily transported into the cells via sugar transporters. Thus, once it was hydrolyzed by β-galactosidase produced inside cells, NO was readily released from the compound. Therefore it took on a novel and efficient approach to deliver RNS （NO） into cells.2. β-Gal-NONOate, which has been reported as a site-specific NO donor with high antibacterial activity（in Chapter 2） is an intriguing agent in therapy and related research. Through this study, β-Gal-NONOate was proved to be promising in overcoming prevalent antibiotic resistance of bacteria. It was applied to get rid of ampicillin resistance of E. coli K-12 with β-galactosidase activity. Bacterial susceptibility to antibiotics was evaluated with the minimum inhibitory concentration （MIC）, which could be measured with E-test After successful inducement of ampicillin-resistant strains, β-Gal-NONOate and NONOate were separately applied to restore antibiotic susceptibility of bacteria through a series of sequential subcultures. The results demonstrated that β-Gal-NONOate possessed greater potential to decrease the MIC of ampicillin-resistant strains than NONOate; while combined with ampicillin for subculture, β-Gal-NONOate was able to kill off the resistant strains within fewer passages than NONOate, that is, to greatly shorten the duration of antibiotic resistance. These results further substantiated that β-Gal-NONOate was a very prospective NO donor, which could be explored and utilized not only as antimicrobial but also as a promising agent to remove increasingly serious antibiotic resistance of bacteria in current times. Based on our previous studies, it is concluded that elimination of antibiotic resistance may be due to more site-specific NO delivery into cells, accessibility of efficaciously intracellular NO level and high efficient antimicrobial roles of β-Gal-NONOate.3. The elucidation of the molecular details of antibiotic resistance will lead to improvements in extending the efficacy of current antimicrobials. Two dimensional electrophoresis（2-DE） is a key technique in studies of proteome. In Chapter 4, proteomic methodologies were applied for the comparative analysis of proteome of E. coli K-12 responded to ampicillin（Amp） resistance and Amp re-susceptivity for understanding of universal pathways that form barriers for antimicrobial agents. For this purpose, three kinds of E. coli K-12（E coli K-12 ancestor strain, E. coli K-12 Amp resistant strain, E. coli K-12 re-susceptible strain） proteome were characterized with the use of 2-DE methods. Then, differential proteins due to Amp resistance were determined by comparing with each other using PDQuest7.2 to study the influence on bacterium in proteome by β-Gal-NONOate. Our findings will be helpful for further understanding of antibiotic-resistant / overcoming resistance mechanism related to proteome. This study also confirmed that β-Gal-NONOate certainly contributes the elimination of Amp by changing the protein expression. 4. So far, nitric oxide （NO） donors have been applied to various aspects of antitumor therapy. To selectively sensitize tumor cells and avoid unwanted side effects, β-Gal-NONOate was firstly used in the cancer research. In this study, we first verified its superiority over its parent NONOate in terms of targeted intracellular NO-releasing and antitumor activity with 9L/LacZ cells in vitro. β-Gal-NONOate only released NO when hydrolyzed by β-galactosidase in 9L/LacZ cells, which led to its more powerful cytotoxicity than that of NONOate. The results showed that β-Gal-NONOate produced higher NO levels than NONOate in 9L/LacZ cells at equal concentration, and hence induced optimal NO levels for antitumor activity. However, in 9L cells, β-Gal-NONOate showed less toxicity than NONOate. Therefore, it is demonstrated that β-Gal-NONOate is a site-specific prodrug for targeting NO intracellularly as a β-galactosidase-sensitive NO donor, and it is also expected to be a promising probe in numerous experimental settings and a potential therapeutic drug for antitumor treatment.5. In order to evaluate the antitumor effects of β-Gal-NONOate in details and to clarify its mechanism to action as an antitumor prodrug, four cell lines were used in Chapter 6. Through cationic liposome-mediated transfection, the eukaryotic expression vector pcDNA3-LacZ was transferred into the HeLa cells; through G418 screening, the HeLa/LacZ cell line steadily expressing β-galactosidase was obtained. With C6/LacZ、 C6、 HeLa/LacZ and HeLa cells as in vitro model, β-galactosidase activities of tumor cells were determined through X-gal staining. NO levels in these four cell lines released from β-Gal-NONOate and NONOate were evaluated with Griess assay. Antitumor effects of NO donors on the four cell lines were estimated by cell counting, cell colony forming rate and MTT （Methylthiazoletetrazolium） assay, respectively. We found that NO levels released from β-Gal-NONOate in C6/LacZ and HeLa/LacZ cells were dependent on its concentration and evidently higher than that from NONOate. And similarly, its antitumor activity to C6/LacZ and HeLa/LacZ cells were obviously more powerful than NONOate（P < 0.05）, but it did not exhibit conspicuous cytotoxicity to C6 cells. However, the NO released from NONOate inside the four cell lines and the effects on all kinds of cells didn’t have great difference. We could draw a conclusion that β-Gal-NONOate was more stable and possessed higher antitumor activity than NONOate. In addition, its action greatly depended on β-galactosidase. It was an effective intracellularly NO release compound, which would be very promising in tumor therapy and relevant research.6. The apoptosis effects of 9L/LacZ, C6/LacZ and HeLa/LacZ cells induced by β-Gal-NONOate were investigated in Chapter 7. Typical apoptotic morphological features included cell shrinkage and condensation and margination of nuclear chromatin were showed by light microscopy. Condensed nuclear chromatin and the morphology of nuclei were demonstrated by fluorescent microscopy with AO and Hoechst 33342 staining. β-Gal-NONOate induced apoptotic DNA breaks were confirmed by a typical "DNA ladder" on agarose gel electrophoresis as well as evidenced by TUNEL assay. β-Gal-NONOate induced apoptosis in a concentration-dependent manner. The apoptotic rates of three cells were measured by flow cytometry with Annexin V-FITC and PI staining. The changes of intracellular [Ca²⁺]_i and mitochondrial transmembrane potential （ΔΨm） were detected using fluorescence indicator Fluo-3/AM and Rhodamine 123 with laster scanning confocal microscopy. β-Gal-NONOate resulted in a rapid increase in [Ca²⁺]_i and a great decrease in ΔΨm. The results of this study confirmed the ability of β-Gal-NONOate to suppress the proliferation of three kinds of cells with typical apoptosis feature in vitro. Disturbance of homeostasis in calcium signaling system might play pivotal roles in apoptosis of three cells. Effects of β-Gal-NONOate were assessed both alone and in paired combination with cisplatin. Q values were used to characterize the interactions as synergistic, additive, or antagonistic. Significant synergistic effects in growth inhinition of β-Gal-NONOate （0.25-10 mM） with cisplatin （2.5-10 μM） on three cell lines were observed （q>1）. We conclude that β-Gal-NONOate may be worth of further studies assessing its value in cancer therapy in combination with the other chemotherapeutic agents.更多还原