【作者】 陈哲；

【导师】 楼宜嘉；

【作者基本信息】 浙江大学 ， 药理学， 2010， 博士

【摘要】 肺癌已成为发病率和死亡率增长最快,严重危害人类健康和生命的恶性肿瘤之一。我国肺癌病死率在城市已居肿瘤死亡首位,非小细胞肺癌(Non-small cell lung cancer,NSCLC)占肺癌的85%,预后较差,致死率高,多数患者确诊时已属晚期。在不同疾病发展阶段需采取不同的治疗策略,早期的局灶性疾病往往用手术治疗而转移性肺癌常采取姑息疗法。药物治疗是控制病程的重要环节,现有药物在有效性方面虽有明显突破,但仍存在毒副反应和多药耐药性问题亟待克服。如何选择高效低毒而不易耐药的药物成为提高疗效的关键。肿瘤耐药和药物毒性依然是临床上肿瘤化疗的难题,这一问题的解决必将对提高肿瘤患者治疗效果、延长生存有重大影响。小分子化合物(small molecular compound)一般由几个或几十个原子组成,药物小分子分子量通常在500以下。按照抗NSCLC的作用原理和来源分类,可包括烷化剂、抗代谢药物、抗肿瘤天然来源小分子、抗肿瘤金属配合物,及酪氨酸激酶抑制剂等等。合成的化学类抗NSCLC药物多数以天然抗肿瘤活性成分为先导化合物研究而来,因此不断发现具有抗肿瘤活性化合物,可为以天然抗癌活性成分为基础,大规模快速筛选先导化合物提供依据。运用计算机模拟和仿生设计对天然产物进行结构修饰改造,是当前及今后一段时期内抗肿瘤药物研究的热点。目前临床常用的抗NSCLC天然来源化合物,主要包括鬼臼毒素、紫杉醇、喜树碱、长春新碱、白藜芦醇、茶多酚等。小分子酪氨酸激酶抑制剂等靶向治疗药物是从分子机理和先导化合物结构出发设计而成,因此,与传统药物相比,对肿瘤细胞的攻击有更好的选择性,具有不良反应相对小,疗效相对高的特点。采用基于结构的靶向设计策略,在设计化合物时引入关于靶标的有价值信息,将会提高活性化合物的命中率,同时降低因合成与筛选大量化合物消耗的成本。相对于传统的药物随机合成研究方法,组合化学提供了结构多样性化合物库构建和丰富的化合物库内化合物样品来源问题。靶向针对表皮生长因子受体(Epidermal growth factor receptor, EGFR)设计喹唑啉化合物库,构建组合化学小分子库,并进行抗NSCLC活性筛选,可望高效快速发现酪氨酸激酶抑制活性化合物,寻找和确认作用多靶点的候选化合物。本论文对若干来源于植物及靶向设计组合化学库内小分子,抗NSCLC活性和机制进行研究,以期找到潜在的更具特异性且效果更好的活性小分子。第一部分补骨脂酚对人非小细胞肺癌A549细胞株增殖抑制作用及机制研究天然来源小分子白藜芦醇(Resveratrol,3,4,5-trihydroxystilbene)是一种很有前景的抗癌药物。白黎芦醇能抑制人类NSCLC细胞增殖,促进细胞凋亡及坏死。补骨脂酚(Bakuchiol)和白藜芦醇的结构相似,两个化合物都具有“(4-羟苯基)-乙烯基(4-hydroxystyryl moiety) ",且均具有抑制DNA聚合酶的作用,该作用可能与两者结构的相似性有关,因此补骨脂酚也极有可能具有抗NSCLC活性。本部分通过对NSCLC A549细胞株增殖、周期变化、胞内活性氧生成、线粒体膜电位(mitochondrial membrane potential,Δψm)变化、细胞凋亡/坏死情况、及相关蛋白表达水平变化等一系列研究,旨在探索补骨脂酚抑制A549增殖活性及其相关机制,并与白藜芦醇进行平行比较。本研究采用MTT法检测补骨脂酚和白藜芦醇对A549细胞体外细胞毒性；用H2-DCFDA染色-流式细胞术(Flow cytometry, FCM)检测细胞内活性氧生成；用JC-1染色-FCM检测线粒体△Ψm变化。用Annexin V/PI双染-FCM、AO/EB染色检测细胞凋亡情况。用PI染色-FCM检测细胞周期变化。用Western-Blotting法检测相关蛋白表达水平变化。实验结果显示,补骨脂酚具有抑制A549细胞增殖活性,作用强于白藜芦醇,补骨脂酚和白藜芦醇作用72 h的ICso分别为9.58±1.12μmol/l和33.02±2.35μmol/l。补骨脂酚作用1h后细胞内ROS显著增加；引起线粒体△Ψm下降,该作用呈量效与时效依赖性,且强于白藜芦醇。补骨脂酚处理36h引起细胞凋亡,而白藜芦醇则以引起细胞坏死为主。补骨脂酚处理48 h后,采用AO/EB进行染色并用荧光显微镜观察,发现出现部分凋亡细胞,核固缩且呈现明亮的红色,呈现明显的量效关系,该作用强于白藜芦醇。此外,周期分布实验表明,补骨脂酚处理24 h引起A549细胞S期阻滞。Western-Blotting结果表明其引起细胞p53、Bax蛋白表达上调,Bcl-2蛋白表达水平下调,出现Caspase 3裂解片段。本实验结果提示,(1)补骨脂酚具有抑制人非小细胞肺癌A549细胞株增殖活性,该作用与经由线粒体依赖通路诱导细胞凋亡,并使细胞周期阻滞相关。(2)补骨脂酚抑制增殖作用明显强于白藜芦醇；其中呈现其特有的诱导ROS-线粒体依赖通路凋亡,可能是两者作用强弱差异的主要机制。第二部分8-芳胺基-3H-咪唑[4,5-g]喹唑啉类衍生物B-2对人非小细胞肺癌A549细胞株体内外抗肿瘤活性及相关机制喹唑啉(quinazoline)杂环是医药化合物中非常重要的核心结构,喹唑啉类衍生物具有重要的生理活性。靶向设计2,3-二取代-8-芳胺基-3H-咪唑[4,5-g]喹唑啉类化合物,通过组合化学构建小分子化合物库,发现新型EGFR酪氨酸磷酸激酶抑制剂是一种崭新的方向。本课题组前期从该化合物库大量具有分子结构多样性化合物中,筛选得到若干抗NSCLC活性化合物[23,24]。其中2-Isopropyl-3-butyl-8-(4-fluorophenylamino)-3H-imidazo [4,5-g] quinazoline (B-2)对人非小细胞肺癌A549细胞株具有最佳增殖抑制活性。本研究第二部分考察了B-2对该细胞株体内外抗肿瘤活性和相关作用机制。本实验首先采用MTT法检测B-2体外对肿瘤细胞和非肿瘤细胞增殖抑制活性。为阐明上述体外抑制增殖作用是否可在体内重现,本研究采用中空纤维内置肿瘤细胞模型,植入实体瘤A549和白血病K562两种不同的细胞株(除了考察体内药物是否分布到达靶部位外,尚评价对A549的选择性抑制作用),给裸鼠口服25、50、100mg/kg的B-2和100mg/kg Iressa(为临床推荐剂量的2.67倍,按体表面积折算),初步探索B-2对裸鼠皮下埋植中空管中细胞增殖抑制活性。之后采用裸小鼠皮下移植瘤模型,评价B-2对荷A549细胞裸小鼠实验治疗作用。并采用计算机辅助受体一配体对接实验、体外激酶活性测试B-2对EGFR酪氨酸激酶活性影响。最后对B-2作用机制进行研究。用PI染色-FCM检测B-2对A549细胞周期分布的影响。用JC-1染色-FCM检测该化合物作用后线粒体ΔΨm变化情况。用Annexin V/PI双染-FCM检测B-2诱导的细胞凋亡。用AO/EB染色观察细胞核损伤情况。用Western-Blotting法检测A549细胞周期和凋亡相关蛋白表达变化。实验结果显示,B-2化合物对A549细胞具有较强体外增殖抑制活性,48 h IC50为4.30±0.33μmol/L。而对非肿瘤来源的细胞EA.hy926和MEF则未呈现明显的抑制增殖作用。中空纤维模型实验中,B-2裸小鼠经口给药,结果发现,化合物B-2特异性地抑制了管内A549细胞的生长,并体现了良好的量效关系,其作用强于Iressa。25、50、100 mg/kg B-2对A549细胞抑制率分别为38.45±2.94%、60.43±1.86%、65.83±2.46%,而100mg/kg Iressa对A549细胞抑制率为47.05±1.15%,但对体重影响不明显。荷A549细胞裸小鼠模型实验中,给药三周后,各组均无一动物死亡,与阴性对照组比较,B-2 50、100、150 mg/kg以及Iressa 100 mg/kg组的小鼠体重无显著变化。B-2 50、100、150 mg/kg组抑瘤率分别为48.69%、85.50%和86.45%,均大于40%,Iressa 100 mg/kg组抑瘤率为67.52%,也大于40%。中剂量组B-2100mg/kg抑瘤作用就显著优于Iressa100 mg/kg。B-2对EGFR酪氨酸激酶活性抑制结果表明,B-2与Iressa的EGFRIC50分别为0.067、0.042μmol/L。计算机辅助进行EGFR蛋白-配基对接实验结果表明,B-2与EGFR具有较好匹配能力,但对接得分弱于Iressa。这与B-2对EGFR酪氨酸激酶活性抑制能力弱于Iressa的结果相吻合。机制研究结果表明,B-2处理24 h引起细胞G1期阻滞,后续Western-Blotting结果表明B-2诱导的G1期阻滞与下调Cyclin D1、CDK4,上调p53、p27Kip1、Rb的蛋白表达水平相关。B-2处理6h即引起A549线粒体△Ψm下降,具有量效依赖关系,提示细胞启动了凋亡程序。B-2处理36h用Annexin V/PI双染-FCM检测1、5、25μmol/L B-2细胞凋亡率分别为6.79±1.11%、14.58±3.53%、50.25±11.34%。B-2处理48 h采用AO/EB进行染色并用荧光显微镜观察,发现出现凋亡细胞,核固缩且呈现明亮的红色,呈现明显的量效关系。后续Western-Blotting结果表明B-2上调Bax/Bcl-2比值,引起细胞色素C大量释放和caspase 9/3激活。提示B-2介导了A549线粒体ΔΨm下降触发的线粒体功能丧失、Bax/Bcl-2比值变化、线粒体细胞色素C释放、caspase活化,最终引起凋亡。本研究结果提示,喹唑啉杂环化合物B-2具有体外选择性抑制人非小细胞肺癌A549增殖作用,作用强于市售同类产品Iressa,但对非肿瘤细胞增殖抑制作用不明显。B-2裸小鼠经口给药,对中空纤维管内A549具有选择性抑制增殖作用；对荷A549细胞裸小鼠具有显著实验治疗作用,作用强于Iressa,但对体重影响不明显。B-2的抗肿瘤作用并非单纯是通过阻断EGFR酪氨酸激酶活性,尚与诱导细胞周期G1期阻滞、促凋亡相关。全文结论：1.补骨脂酚具有抑制人非小细胞肺癌A549细胞株增殖活性,该作用与经由线粒体依赖通路诱导细胞凋亡,并使细胞周期阻滞相关。补骨脂酚抑制增殖作用明显强于白藜芦醇；其中呈现其特有的诱导ROS-线粒体依赖通路凋亡,可能是两者作用强弱差异的主要机制。2.喹唑啉杂环化合物B-2具有体外选择性抑制人非小细胞肺癌A549增殖作用,作用强于市售同类产品Iressa,但对非肿瘤细胞增殖抑制作用不明显。B-2裸小鼠经口给药,对中空纤维管内A549具有选择性抑制增殖作用；对荷A549细胞裸小鼠具有显著实验治疗作用,作用强于Iressa,但对体重影响不明显。B-2的抗肿瘤作用并非单纯是通过阻断EGFR酪氨酸激酶活性,尚与诱导细胞周期G1期阻滞、促凋亡相关。更多还原

【Abstract】 The morbidity and mortality of the lung cancer has become the fastest-growing in a variety of cancers, which seriously harm to human’s health and life. China’s mortality rate of lung cancer in the city now ranks first in cancer mortality. Non-small cell lung cancer (NSCLC) accounts for 85% of lung cancer patients, associated with poor prognosis and high death rate.The majority of patients belonged to the later period of caancer process when diagnosed. At different stages of development of the disease should be taken to a different treatment strategies.Surgical treatment aimed at early-stage of focal disease, while palliative treatment targeted of metastatic lung cancer. Drug therapy is one of the pillars of its treatment. The key point in enhancing the effect of treatment may be how to choose a relatively sensitive drug to avoid drug resistance.Tumor resistance and drug toxicity remains a problem in clinical cancer chemotherapy. Resolution of this problem will certainly improve the treatment of cancer patients and extend the life of patients.Small molecular compounds are usually formed by several or dozens of atoms, whose molecular weight are in the dozens to hundreds (general molecular weight below 500). In accordance with the role of anti-NSCLC principle and source categories, small molecular compounds may include alkylating agents, anti-metabolism drugs, natural source of anti-tumor small molecules, anti-tumor metal complexes, as well as small molecular tyrosine kinase inhibitors and so on.The research and development of most synthetic chemical anti-NSCLC drugs are based on natural active ingredients as the leading compound. Based on natural active anti-cancer ingredients, large-scale, rapid screening of leading compounds and using computer simulation and bionic design to carry out structural modification of the transformation of natural products will be a hotspot of antitumor drugs during the current and future period of time. Anti-NSCLC chemotherapy herbal and complementary medicine which are commonly used in the current clinical treatment include podophyllotoxin, paclitaxel, camptothecin, vincristine, resveratrol, polyphenols and so on.Small molecular tyrosine kinase inhibitors which used in targeted therapy are developed from the molecular mechanism. Therefore, compared with traditional drugs, they have better selectivity and fewer side effects.Using structure-based targeted design strategy to design compounds will increase the hit rate of active compounds and reduce the cost of the synthesis and screening of a large number of compounds。Opposed to the traditional drug discovery methods based on natural leading compounds, emerging combinatorial chemistry efficiently resolved the problem of the origins of the samples. Designing dioxane quinazoline compound libraries as a new EGFR tyrosine kinase inhibitor libraries, using combinatorial chemistry and anti-NSCLC pharmacological screening, will comply rapid and efficient discovery of active anti-NSCLC compounds.In this paper, anticancer activity and mechanism of several small molecules (from plants and target-designed combinatorial chemistry libraries) on non-small cell lung cancer A549 was studied, hoping to find more potential and specific small molecules.1 Anti-tumor effects of bakuchiol and mechanism on human lung adenocarcinoma A549 cell lineResveratrol (3,5,4-trihydroxy-trans-stilbene), a phytoalexin found in grapes, berries, and peanuts, is one of the most promising agents for cancer prevention.In view of the anti-NSCLC potential of resveratrol,it is expected to discover novel chemotherapeutic agents with resveratrol-like structure. Both of bakuchiol and resveratrol have a "4-hydroxystyryl moiety", which suggests that bakuchiol may also play an anti-tumor activity.Therefore, the antitumor activity of bakuchiol, an analogue of resveratrol, was explored on human lung adenocarcinoma cells.MTT assay revealed that IC50 of bakuchiol at 72 h was 9.58±1.12μmol/1,much lower than that of resveratrol (33.02±2.35μmol/1). Bakuchiol but not resveratrol elevated intracellular reactive oxygen species (ROS). Bakuchiol reduced mitochondrial membrane potential (Δψm) of cells in a concentration-and time-dependent manner, showing more potent effect than that of resveratrol.More apoptotic cells were induced by bakuchiol, compared with resveratrol. Subsequently, S-phase arrest, caspase 9/3 activaton, p53 and Bax up-regulation, as well as Bcl-2 down-regulation were observed in bakuchiol-treated A549 cells. The results point toward that S phase-related cell cycle regulation, more importantly ROS-related apoptosis might contribute to the anticancer properties of bakuchiol, which will strongly support the further development of bakuchiol against NSCLC.These results suggested that bakuchiol has selective cytotoxic activity on human lung adenocarcinoma A549 cell line but has hardly any cytotoxicity in other nontumorous cell lines. Bakuchiol showed more potent anti-tumor effect on A549 cells than that of resveratrol. S phase-related cell cycle regulation, more importantly ROS-related apoptosis might contribute to the anticancer properties of bakuchiol.2 Anti-tumor effects and mechanism of 2,3-disubstituted 8-arylamino-3H-imidazo [4,5-g] quinazoline derivative B-2 on human lung adenocarcinoma A549 cell line in vitro and in vivoQuinazoline heterocyclic compounds is a very important medicine core structure. Quinazoline derivatives have important biological activities. Chemists targeting designed 2,3-disubstituted 8-arylamino-3H-imidazo [4,5-g] quinazolines compound libraries as a new EGFR tyrosine kinase inhibitor libraries and constructed small molecule combinatorial chemistry library. Previously we screened the anti-cancer activity of these compounds on different cancer cell lines. Among them, B-2 (2-Isopropyl-3-butyl-8-(4-fluorophenylamino)-3H-imidazo [4,5-g] quinazoline possessed high anti-NSCLC activity.In this part, the antitumor effect of B-2 (compared with Iressa) against human lung adenocarcinoma A549 cell line in vitro and in vivo were investigated.Cytotoxicity assays revealed that B-2 was a potential antitumor compound with IC50 value of (4.30±0.30)μmol/L at 48 h in human lung adenocarcinoma A549 cell line. The MTT assay and morphological analysis revealed a remarkable difference between B-2-treated A549 and nontumorous cells.In hollow fiber, containing two different tumor cell lines, B-2 selectively inhibited A549 cells and was demonstrated strong antitumor activity with a dose-dependent effect in vivo. The cell inhibition rates against A549 cells in hollow fiber were (38.45±2.94)%, (60.43±1.86)% and (65.83±2.46)% respectively, at the doses of B-225,50 and 100 mg/kg; while cell inhibition rate of 100 mg/kg Iressa was (47.05±1.15)%.In the study with A549 xenograft models, B-2 was administrated orally once a day for five 5 days/wk for 3 weeks. The inhibition rates were 48.69%,85.50% and 86.45%, respectively, at the doses of B-2 50,100 and 150 mg/kg. The treatment effect of B-2 is better than Iressa at the same dose (100 mg/kg). No significant effect on body weight of nude mice was observed after B-2 treatment.However, B-2 exhibited weaker epithelial growth factor receptor (EGFR) ligand-binding affinity compared with its parent compound Iressa according to the docking results and EGFR PTK assay. Administration of B-2 for 24 h caused a concentration dependent increase in the proportion of cells in the G1 phase in comparison with control cultures. B-2 increased the protein levels of p53, p27K1P1 and pRb while decreased the expression of cyclin-dependent kinase 4 (CDK4) and cyclin Dl.After 6 h treatment,1～25μmol/L B-2 lowered mitochondrial transmembrane potential presented as green fluorescence in a dose-dependent manner, indicating a dose-dependent decrease of the mitochondrial transmembrane, proved that B-2-induced early stage depolarization of the mitochondrial transmembrane followed apoptosis. After incubation with B-2 for 36 h, demonstration of apoptosis was shown by annexin V and propidium Iodide staining for apoptosis. The apoptotic rates were (6.79±1.11)%, (14.58±3.53)% and (50.25±11.34)%, respectively, at the concentrations of B-2 1,5 and 25μmol/L. Then after 48 h treatment of B-2, AO-EB staining showed a number of cells exhibited a flattened polygonal morphology, and partial cells showed morphological features of apoptosis.In addition, B-2-induced death of A549 cells were identified by characteristics of apoptosis including Cytochrome C release and huge increase of Bax/Bcl-2 ratio. These events were accompanied by activation of caspase-3 and-9.These results suggested that B-2 demonstrated strong antitumor activity with a dose-dependent effect in vitro and in vivo on human lung adenocarcinoma A549 cell line. Inhibition of cell cycle progression and induction of apoptotic cell death contributed to the anti-NSCLC effects of B-2.Summary1. Bakuchiol has selective cytotoxic activity on human lung adenocarcinoma A549 cell line but has hardly any cytotoxicity in other nontumorous cell lines. Bakuchiol showed more potent anti-tumor effect on A549 cells than that of resveratrol. S phase-related cell cycle regulation, more importantly ROS-related apoptosis might contribute to the anticancer properties of bakuchiol.2. B-2 demonstrated strong antitumor activity with a dose-dependent effect in vitro and in vivo on human lung adenocarcinoma A549 cell line. Inhibition of cell cycle progression and induction of apoptotic cell death contributed to the anti-NSCLC effects of B-2.更多还原